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Trace heavy metal ions promoted extracellular electron transfer and power generation by Shewanella in microbial fuel cells
Bioresource Technology 211 (2016) 542–547
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Trace heavy metal ions promoted extracellular electron transfer and power generation by Shewanella in microbial fuel cells Yu-Shang Xu a,b,1, Tao Zheng c,1, Xiao-Yu Yong b, Dan-Dan Zhai a, Rong-Wei Si a, Bing Li a, Yang-Yang Yu a, Yang-Chun Yong a,⇑ a b c
Biofuels Institute, School of the Environment, Jiangsu University, Zhenjiang 212013, China College of Biotechnology and Pharmaceutical Engineering and Bioenergy Research Institute, Nanjing TECH University, Nanjing 210095, China Guangzhou Institute of Energy Conversion, Key Laboratory of Renewable Energy, Chinese Academy of Science, Guangzhou, Guangdong 510640, China
h i g h l i g h t s 2+ or Cd2+. or Cd2+ improved riboflavin accumulation and anodic bacterial attachment. Enhanced EET might be the underlying mechanism for MFCs performance improvement. Trace heavy metal ions addition is a practical way to improve MFCs performance.
MFCs performance was improved by addition of trace level of Cu
Cu
2+
a r t i c l e
i n f o
Article history: Received 11 February 2016 Received in revised form 24 March 2016 Accepted 25 March 2016 Available online 28 March 2016 Keywords: Microbial fuel cells Shewanella oneidensis Heavy metal ions Extracellular electron transfer Bioelectrochemical systems Biofilm
a b s t r a c t Although microbial fuel cells (MFCs) is considered as one of the most promising technology for renewable energy harvesting, low power output still accounts one of the bottlenecks and limits its further development. In this work, it is found that Cu2+ (0.1 lg L 1–0.1 mg L 1) or Cd2+ (0.1 lg L 1–1 mg L 1) significantly improve the electricity generation in MFCs. The maximum power output achieved with trace level of Cu2+ (6 nM) or Cd2+ (5 nM) is 1.3 times and 1.6 times higher than that of the control, respectively. Further analysis verifies that addition of Cu2+ or Cd2+ effectively improves riboflavin production and bacteria attachment on the electrode, which enhances bacterial extracellular electron transfer (EET) in MFCs. These results unveil the mechanism for power output enhancement by Cu2+ or Cd2+ addition, and suggest that metal ion addition should be a promising strategy to enhance EET as well as power generation of MFCs. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Microbial fuel cells (MFCs) are promising for sustainable wastewater treatment as it perfectly integrates pollutants treatment with energy generation (Logan and Rabaey, 2012; Schroder, 2011). They also showed valuable potential on a variety of environmental and biochemical applications such as biosensor (Yang et al., 2015a), hydrogen production (Cheng and Logan, 2007), and even as remote/low density power supply (Donovan et al., 2011). MFCs characterize itself with other fuel cell technologies by adopting live electrochemically active bacterium (EAB), like Geobacter, Shewanella and Pseudomonas, as biocatalyst to facilitate energy conversion by oxidization the substrate in central metabolism ⇑ Corresponding author. 1
E-mail address: [email protected] (Y.-C. Yong). Equal contribution.
http://dx.doi.org/10.1016/j.biortech.2016.03.144 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
and transfer the released electron to the external circuit (Lovley, 2008). Most of the bacteria are able to convert chemical energy to intracellular electrons, but only EAB has the specific pathways to transfer intracellular electrons to extracellular electrode (Logan, 2009). Thus, electron transfer between bacteria and electrode which termed as extracellular electron transfer (EET) is the unique characteristic of EAB, and EET efficiency is one of the most important topics in the field of MFCs. Therefore, versatile strategies have been developed to improve EET efficiency and the energy output capability of MFCs. Most of these efforts are mainly targeted on electrode modification (Liao et al., 2015), novel anode structure design (Qiao et al., 2008) and bioreactor configuration (Logan et al., 2015). Recently, enhancement of EET efficiency with mutualistic interaction between microorganisms (Wang et al., 2015; Yang et al., 2015c), addition of conjugated oligoelectrolyte (Zhao et al., 2015), genetic engineering (Liu et al., 2012; Yang et al., 2015b; Yong et al.,
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2014a) and environmental factors manipulation (Fitzgerald et al., 2012; Yong et al., 2012) gained increasing attention as they successfully proved their value in efficiency improvement from ‘‘biological” side. Among these achievements, environmental factor (pH, metal ions, etc.) manipulation was considered to be one of the most promising approaches due to low cost and ease to operation. Electrolyte pH is an important parameter that affects the performance of MFCs. It was found that some EAB could take advantages of alkaline electrolyte (Yong et al., 2013; Yuan et al., 2011), while others preferred to acidic circumstance (Nimje et al., 2011). Thus, optimization of electrolyte pH is necessary and could significantly improve the performance of MFCs (Zhuang et al., 2010). Metal ions such as Fe3+, Ca2+, and Na+ were also explored to stimulate energy output of MFCs. Addition of Fe3+ (10 mM) increased the maximum power output of MFCs by 1.1 times (Wu et al., 2013), while Ca2+ addition (1.4 mM) enhanced the current output of MFCs by 80% (Fitzgerald et al., 2012). In addition, Na+ (20 g L 1) improved the maximum power output of MFCs by 30% (Lefebvre et al., 2012). These results suggested that pH optimization and metal ion addition would be effective ways to improve MFCs performance. However, adjust pH of bulk electrolyte is still cost intensive, and sometimes may be unavailable. Addition of metal ions would be more feasible in practical application. Heavy metal ions usually showed sophisticated effects on bacteria depends on their concentration, e.g., they are usually detrimental to bacteria at high concentration, while some of them might induce/stimulate the cellular metabolism at low concentration (Paraszkiewicz et al., 2007; Xu et al., 2014). However, the effects of heavy metal ions on EET and MFCs performance are still unclear. In this study, the effects of heavy metal ions on energy output of MFCs were investigated. Interestingly, it was found that nanomolar Cu2+ or Cd2+ significantly improved power output of MFCs by increasing electron shuttle accumulation and biofilm formation. The results provided valuable knowledge for MFCs operation and suggested trace heavy metal ion addition would be a practical approach for efficient EET and enhancement on energy output of MFCs.
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2.2. Bacteria and growth condition Shewanella oneidensis MR-1 was cultured at 30 °C in LB broth with shaking at 200 rpm until the optical density at 600 nm (OD600) reached about 5.0. The cells were collected by centrifugation (5000 rpm, 5 min) and washed with M9 for three times. Then, the cell pellets were re-suspended with electrolyte (95% M9 medium and 5% LB broth, 18 mM lactate) and were adjusted to the desired cell concentration (OD600 = 2.5), and were inoculated into the anodic chamber of the MFCs (30 ml). The effects of metal ions on the MFCs performance were tested by changing the metal ions’ final concentration. Each test was conducted in batch mode. Metal ions used in this study are copper (II) (CuSO4), cadmium (II) (CdCl2), zinc (II) (ZnCl2), chromium (III) (CrCl3), manganese (II) (MnCl2), cobalt (II) (CoCl2), nickel (II) (NiSO4). Stock solution (1 g L 1) of each metal ion was prepared with electrolyte and stored at 4 °C until use. For addition of metal ions, 300 lL of diluted (the times of dilution was dependent on the final concentration needed for MFCs) metal ion solution was immediately injected into the anodic chamber of MFCs after bacteria inoculation. Each metal ion and concentration is tested independently in batch mode, and all experiments are conducted in triplicates. 2.3. HPLC analysis of riboflavin The concentration of riboflavin was analyzed by using reverse phase high performance liquid chromatography (HPLC) according to the reported method (Yong et al., 2013). In brief, 2 ml of anodic culture suspension from MFCs was centrifuged (5000 rpm 5 min), and the supernatant was filtrated with 0.22 lm cellulose membrane and then subjected to HPLC analysis. HPLC analysis was performed at a Shimadzu HPLC system (Japan) equipped with a UV detector. The column used was a Shimadzu C18 analytical column (5 micro-m particle size, 250 mm 4.6 mm i.d.). Riboflavin was separated in a solvent system of water–methanol–acetic acid (68:32:0.1 v/v) and a flow rate of 1.0 mL min 1, the HPLC profile was monitored at 254 nm. 2.4. Quantitative biofilm detection
2. Materials and methods 2.1. MFCs set-up and electrochemical measurements Dual chamber MFCs (the size of each chamber being 3 cm (width) 5 cm (length) 6 cm (height)) separated with nafion 117 membrane (Dupont, USA) was used in this work (Yong et al., 2013, 2014b). Carbon cloth with size if 2 cm 3.5 cm and 1 cm 2 cm were used as cathode and anode electrodes, respectively. Saturated calomel electrode (SCE) electrode (CH Instrument, Shanghai, China) was inserted into each chamber as reference electrode when needed. The anodic medium was M9 medium supplemented with 18 mM sodium lactate as carbon source and electron donor (Yong et al., 2012, 2014b). The cathodic solution was 50 mM K3[Fe(CN)6] in 50 mM KCl solution. For current generation measurement, a 2 KX external resistor was connected into the circuit of MFCs, and the potential of the MFCs was recorded by a digital multimeter. Current density (I) was calculated as I = V (output voltage)/R (external resistance)/A (anode surface area), and power density (P) was calculated as P = V I. All other electrochemical measurements were conducted using CHI 660E electrochemical working station (CH Instrument, Shanghai, China). Power output and polarization curves were obtained by varying the external loading resistor (Qiao et al., 2008). The bacteria attachment on the anodes was observed with scanning electron microscopy (SEM) (JEOL, JSM-7001F, Japan).
Quantitative biofilm formation ability assay was conducted based on a crystal violet staining method (Morales-Calderon et al., 2012) with slight modification. Briefly, strain S. oneidensis MR-1 was cultured with shaking (200 rpm) in LB medium for 15 h at 30 °C. Then, 1 ml of culture suspension was centrifuged (5000 rpm 5 min). Then the cell pellets were re-suspended with 2 ml fresh medium (95% M9 medium and 5% LB broth, 18 mM sodium lactate). After that, 200 lL of cell suspensions were added into each well of 96-well plate. The plate was incubated at 30 °C for another 48 h. After that, planktonic cells were removed. The surface-associated biomass was stained by 0.5% crystal violet for 15 min. Then, the stained biofilm was dissolved with 200 lL of 95% ethanol, and the amount of biofilm biomass was quantified by A595 with a microplate reader (Tecan, Männedorf, Switzerland). 3. Results and discussion 3.1. Effect of metal ions on performance of MFCs Metal ions, in particular heavy metal ions, have marked impacts on the activity of microbes, which will affect the performance of MFCs as expected. The voltage outputs of MFCs with the addition of different metal ions were measured. As listed in Table 1, all the metal ions tested except Cd2+ exhibited inhibition on the voltage outputs of MFCs at certain concentration, which might due to
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Table 1 Effect of different metal ions on the voltage output of MFCs* and the minimum inhibitory concentration (MIC) for Cu2+ or Cd2+. Concentration 1 lg L 100 lg L 1 1 mg L 1 10 mg L 1 MIC (mg L 1) 1
Cu2+
Cd2+
Zn2+
Cr3+
Mn2+
**
+ + + + 300
+ +
+ +
+ + +
+ +
800
***
NT
NT NT
NT
Co2+
Ni2+
NT NT NT NT
NT NT NT NT
*
Each condition was tested independently at batch mode. + Indicates enhancement; + indicates no significant effect; repression. *** NT, not tested. **
indicates
their toxic effect on S. oneidensis MR-1. For example, Co2+ and Ni2+ showed significant inhibition at the concentration of 1 lg L 1. As these heavy metal ions are toxic to bacteria, it is normal to expect these inhibition phenomena. Surprisingly, Cu2+ and Cd2+ enhanced the voltage output of MFCs significantly, once the concentration is lower than 100 lg L 1 and 1 mg L 1, respectively (Table 1). This unexpected exploration is interesting as it might be used to improve the MFCs performance. Thus, the effects of Cu2+ and Cd2+ on MFCs performance were systematically investigated. To get the highest energy output of MFCs, the addition concentrations of these two heavy metal ions were optimized. As shown in Fig. 1, the maximum voltage outputs of MFCs with the addition of Cu2+ or Cd2+ at different concentration were monitored. Compared to the control (without addition) with the maximum voltage output of 117 ± 5 mV, supplemented with Cu2+ and Cd2+ showed great enhancement of bioelectricity generation under the concentration lower than 100 lg L 1. In the assay supplemented with Cu2+, the highest voltage output of 165 ± 6 mV was achieved at the concentration of 1 lg L 1, which was 1.4 times of the control. Similarly, for Cd2+, it was found that the voltage output was enhanced when the concentration is lower than 1 mg L 1. The highest voltage output of 175 ± 8 mV was obtained with the addition of 1 lg L 1 Cd2+, which was 1.5 times of the control. Previous research showed that Fe3+ (6 mM) supplementation in S. oneidensis MR-1 inoculated MFCs increased the electricity generation (Wu et al., 2013). In addition, Fitzgerald et al. found that addition of CaCl2 (>300 lM) increased the current density by >80% in S. oneidensis MR-1 inoculated MFCs (Fitzgerald et al., 2012). These findings suggested that addition of metal ions in MFCs was an effective way for performance improvement. However, addition of heavy metal ions in MFCs is seldom studied due to high concentration of heavy metal ions are detrimental to cells and might result in water pollution. Interestingly, this study found that heavy metal ions (Cu2+ or Cd2+) at the concentrations 105 times lower than their the minimum inhibitory concentrations (MIC) also significantly improved MFCs performance. Thus, the finding of this study highlighted that heavy metal ions addition would also be another promising and practical for improvement of MFCs performance.
Fig. 1. Effect of Cu2+ or Cd2+ on the maximum voltage output of MFCs. Each condition was tested independently at batch mode. Three replicates are performed for each experiment.
3.2. Enhancement of MFCs performance by Cu2+ and Cd2+ addition Then, the effect of Cu2+ or Cd2+ on MFCs performance was investigated in details, respectively. As shown in Fig. 2a, the voltage output of MFCs steadily increased after bacteria inoculation and reached its maximum value of 117 mV for MFCs without heavy metal addition at the first cycle. For MFCs supplemented with Cu2+ or Cd2+, the voltage outputs increased faster than that of the control, and higher maximum values were obtained (about 169 ± 6 mV for Cu2+ addition, and about 175 ± 8 mV for Cd2+ addition). At the following cycles, the reproducible voltage output was obtained by feeding fresh sodium lactate as electron donor. The
Fig. 2. Performance of MFCs supplemented with different metal ions. (a) Timecourse profiles of voltage outputs (MFC with a 2 KX exterior resistor) of MFCs with (1 lg L 1 Cu2+ or Cd2+) or without (control) metal ions addition. Arrows indicates lactate (final concentration, 18 mM) was added. (b) Power output and polarization curves of MFCs with (1 lg L 1 Cu2+ or Cd2+) or without (control) metal ions addition.
polarization and power output curves, which were obtained by varying external loading resistance, were plotted to investigate the effect of Cu2+ or Cd2+ on the power output of MFCs (Fig. 2b). The maximum power density of the MFCs supplemented with Cu2+ was 92.4 mW m 2, which was 1.3 times higher than that of
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the control (39.8 mW m 2). Similarity, The maximum power density of the MFCs supplemented with Cd2+ was 104.4 mW m 2, which was 1.6 times higher than that of the control. Meanwhile, it was found that the open circuit voltage (OCV) of the MFCs supplemented with Cu2+ (676 mV) or Cd2+ (673 mV) was higher than that of the control (579 mV), which might favor EET and power output. These results confirmed that trace amount of Cu2+ and Cd2+ could improve the energy output of S. oneidensis MR-1 inoculated MFCs. Some metal ions may serve as trace element for bacteria to improve cell growth at low concentration, which might in turn enhance MFCs performance. Thus, the effect of Cu2+ or Cd2+ on cell growth of S. oneidensis MR-1 was further investigated. At high concentration (10 mg L 1), both of Cu2+ and Cd2+ showed slight growth inhibition (Fig. S1). However, according to the cell growth curves, there was no significant difference between the control and that with trace level (1 lg L 1, 105 times lower than MIC) of Cu2+ or Cd2+ addition. The results excluded the possibility of cell growth promotion related MFCs performance improvement by Cu2+ or Cd2+, and suggested that the power output improvement by Cu2+ or Cd2+ addition should be ascribed to other factors besides cell growth. 3.3. Increased riboflavin production by Cu2+ and Cd2+ addition Extracellular electron transfer (EET) efficiency was considered to be the limiting parameter that determined the performance of MFCs (Yang et al., 2012). To date, there are two EET pathways have been identified for EAB, i.e., direct electron transfer through outer membrane redox active proteins (e.g., c-type cytochromes) or conductive nanowire, and indirect electron transfer mediated by exogenous or endogenous electron shuttles (e.g., pyocyanin, riboflavin) (Lovley, 2006; Yang et al., 2012). S. oneidensis MR-1 usually employed c-type cytochrome for direct EET and indirect EET mediated by electron shuttle of riboflavin (Marsili et al., 2008; Yong et al., 2014b). To test the effect of metal ions on EET of S. oneidensis MR-1 in MFCs, cyclic voltammetry (CV) was conducted when the voltage outputs reached the steady state. As illustrated in Fig. 3a, MFCs with or without metal ions showed clear catalytic wave with mid-point potential 0.42 V (vs SCE), which was in agreement with the electrochemical reaction of riboflavin (the main electron shuttle of S. oneidensis MR-1) (Yong et al., 2013). Interestingly, the catalytic currents of CV obtained from MFCs with heavy metal ions addition (regardless of Cu2+ or Cd2+) (j2 shown in Fig. 3a) were much higher than that of the control (j1 shown in Fig. 3a). The results substantiated that trace level of heavy metal ions improved electron shuttle mediated EET, which might contribute to the enhancement of MFCs power output. It also implied that heavy metal ions might induce overproduction of riboflavin in S. oneidensis MR-1. The concentration of riboflavin in MFCs was further analyzed by HPLC after MFCs discharge. As shown in Fig. 3b, riboflavin concentrations detected in the MFCs with Cu2+, or Cd2+ are 0.67 ± 0.02, and 0.65 ± 0.01 lg mL 1, respectively, which were 21.8%, and 18.2% higher than that of the control (0.55 ± 0.01 lg mL 1). The result confirmed the CV analysis and indicated that heavy metal ions improved riboflavin production of S. oneidensis MR-1. Heavy metal ions including Cu2+ and Cd2+ are usually used as inducers to improve the production of secondary metabolites by plants, fungi or bacteria (Maksymiec et al., 2005; Morgenstern et al., 2015; Xu et al., 2014). For example, Cu2+ or Cd2+ addition induced the accumulation of jasmonates in mature leaves of Arabidopsis thaliana (Maksymiec et al., 2005). Co2+ was also proved to be an effective inducer on antibiotic production by Streptomyces coelicolor (Morgenstern et al., 2015). Here, this study demonstrated that Cu2+ or Cd2+ at trace level could induce enhancement on
Fig. 3. (a) Cyclic voltammograms (CV) of MFCs with (1 lg L 1 Cu2+ or Cd2+) or without (control) metal ions addition. ‘‘j1” and ‘‘j2” indicates the catalytic current of the corresponding CV profiles, respectively. (b) Riboflavin concentration in MFCs with (1 lg L 1 Cu2+ or Cd2+) or without (control) metal ions addition analyzed by HPLC. Three replicates are performed for each experiment.
riboflavin (a typical secondary metabolite of bacteria) production (Fig. 3b). As riboflavin is the major endogenous electron shuttle of S. oneidensis MR-1 that mediated the indirect EET between bacteria and electrode, the improved riboflavin production underlies the enhancement of energy output by heavy metal ions. 3.4. Enhanced anodic bacterial attachment by Cu2+ and Cd2+ addition Anodic biofilm, as a bacterial community adhered to the anode of MFC, also played important roles for power generation in MFCs. Better anodic biofilm formation usually followed with higher power output (Yu et al., 2011). Increase of biofilm formation is a common protective mechanism for bacteria when exposed to heavy metal ions, as biofilm can withstand higher toxicity (Morales-Calderon et al., 2012). The sub-lethal level of toxic chemicals may induce extensive synthesis of extracellular polysaccharide and enhancement on c-di-GMP signaling, which in turn improve the biofilm formation (Chua et al., 2015). Thus, the formation of anodic biofilm under different conditions was observed by SEM after MFCs discharge. As shown in Fig. S2, there are only a few bacteria attached on the surface of the anodic electrode due to poor biofilm formation ability of S. oneidensis MR-1. However, after Cu2+ addition, much more cells attached on the surface of the electrode were observed. The increased bacteria attachment was also observed after Cd2+ addition.
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51578266, 21306069), Natural Science Foundation of Jiangsu Province (BK20130492), Specialized Research Fund for the Doctoral Program of Higher Education (Ministry of Education, 20133227120014).
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.biortech.2016.03. 144. References
Fig. 4. Quantitative analysis of biofilm formation ability of S. oneidensis MR-1 (control, 1 lg L 1 Cu2+ and 1 lg L 1 Cd2+). At least five replicates are performed for each experiment.
To further confirm the SEM observation, another biofilm formation assay was conducted. In this assay system, the biofilm biomass formed on the wall of 96 micro-wells were stained and quantified. As shown in Fig. 4, the biofilm formation ability of S. oneidensis MR-1 was significantly enhanced by Cu2+ or Cd2+ addition. Compared with the control without any addition, the biofilm formation of S. oneidensis MR-1 was increased about 45% with Cu2+ addition, while it increased about 29% with Cd2+ addition. Although the electrodes (carbon fiber) and 96 micro-wells (polypropylene) are made from different material, Cu2+ or Cd2+ addition could increase the bacteria attachment on the surface of both materials. The results suggested Cu2+ and Cd2+ might induce a general pathway to enhance the bacterial attachment on abiotic surface. The results implied that enhancement of bacteria attachment on the anodic electrode by heavy metal ions might also contribute to the power output improvement. It is interesting that there are much more cells attached on the electrode surface with Cu2+ addition than that with Cd2+ addition (Fig. S2), while the power output of MFCs under these two different conditions was nearly the same (Fig. 2). The results implied that enhanced anodic bacteria attachment and electron shuttle concentration might just partially contributed to the power output improvement, other factors related to power output such as cellular metabolism and activity of electron transfer chain (e.g., c-type cytochrome) might be affected by Cu2+ or Cd2+ and might play a role for energy output improvement. 4. Conclusion This study showed an interesting concentration-dependent effects of heavy metal ions on the performance of MFCs. Impressively, Cu2+ and Cd2+ at trace level (5–6 nM) significantly improved MFCs performance, i.e., 1.3–1.6 times higher power output was obtained compared to that of the control. Further analyses indicated that enhancement on electron shuttle synthesis (riboflavin) and anodic biofilm formation was the underlying mechanism for heavy metal ions improved EET as well as MFCs performance. These results provided mechanistic insights into the effect of heavy metal ions on the performance of MFCs, which is valuable for practical operation of MFCs. Acknowledgements The authors sincerely acknowledge the financial supports from National Natural Science Foundation of China (NSFC grant Nos.
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Trace heavy metal ions promoted extracellular electron transfer and power generation by Shewanella in microbial fuel cells Yu-Shang Xu a,b,1, Tao Zheng c,1, Xiao-Yu Yong b, Dan-Dan Zhai a, Rong-Wei Si a, Bing Li a, Yang-Yang Yu a, Yang-Chun Yong a,⇑ a b c
Biofuels Institute, School of the Environment, Jiangsu University, Zhenjiang 212013, China College of Biotechnology and Pharmaceutical Engineering and Bioenergy Research Institute, Nanjing TECH University, Nanjing 210095, China Guangzhou Institute of Energy Conversion, Key Laboratory of Renewable Energy, Chinese Academy of Science, Guangzhou, Guangdong 510640, China
h i g h l i g h t s 2+ or Cd2+. or Cd2+ improved riboflavin accumulation and anodic bacterial attachment. Enhanced EET might be the underlying mechanism for MFCs performance improvement. Trace heavy metal ions addition is a practical way to improve MFCs performance.
MFCs performance was improved by addition of trace level of Cu
Cu
2+
a r t i c l e
i n f o
Article history: Received 11 February 2016 Received in revised form 24 March 2016 Accepted 25 March 2016 Available online 28 March 2016 Keywords: Microbial fuel cells Shewanella oneidensis Heavy metal ions Extracellular electron transfer Bioelectrochemical systems Biofilm
a b s t r a c t Although microbial fuel cells (MFCs) is considered as one of the most promising technology for renewable energy harvesting, low power output still accounts one of the bottlenecks and limits its further development. In this work, it is found that Cu2+ (0.1 lg L 1–0.1 mg L 1) or Cd2+ (0.1 lg L 1–1 mg L 1) significantly improve the electricity generation in MFCs. The maximum power output achieved with trace level of Cu2+ (6 nM) or Cd2+ (5 nM) is 1.3 times and 1.6 times higher than that of the control, respectively. Further analysis verifies that addition of Cu2+ or Cd2+ effectively improves riboflavin production and bacteria attachment on the electrode, which enhances bacterial extracellular electron transfer (EET) in MFCs. These results unveil the mechanism for power output enhancement by Cu2+ or Cd2+ addition, and suggest that metal ion addition should be a promising strategy to enhance EET as well as power generation of MFCs. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Microbial fuel cells (MFCs) are promising for sustainable wastewater treatment as it perfectly integrates pollutants treatment with energy generation (Logan and Rabaey, 2012; Schroder, 2011). They also showed valuable potential on a variety of environmental and biochemical applications such as biosensor (Yang et al., 2015a), hydrogen production (Cheng and Logan, 2007), and even as remote/low density power supply (Donovan et al., 2011). MFCs characterize itself with other fuel cell technologies by adopting live electrochemically active bacterium (EAB), like Geobacter, Shewanella and Pseudomonas, as biocatalyst to facilitate energy conversion by oxidization the substrate in central metabolism ⇑ Corresponding author. 1
E-mail address: [email protected] (Y.-C. Yong). Equal contribution.
http://dx.doi.org/10.1016/j.biortech.2016.03.144 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
and transfer the released electron to the external circuit (Lovley, 2008). Most of the bacteria are able to convert chemical energy to intracellular electrons, but only EAB has the specific pathways to transfer intracellular electrons to extracellular electrode (Logan, 2009). Thus, electron transfer between bacteria and electrode which termed as extracellular electron transfer (EET) is the unique characteristic of EAB, and EET efficiency is one of the most important topics in the field of MFCs. Therefore, versatile strategies have been developed to improve EET efficiency and the energy output capability of MFCs. Most of these efforts are mainly targeted on electrode modification (Liao et al., 2015), novel anode structure design (Qiao et al., 2008) and bioreactor configuration (Logan et al., 2015). Recently, enhancement of EET efficiency with mutualistic interaction between microorganisms (Wang et al., 2015; Yang et al., 2015c), addition of conjugated oligoelectrolyte (Zhao et al., 2015), genetic engineering (Liu et al., 2012; Yang et al., 2015b; Yong et al.,
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2014a) and environmental factors manipulation (Fitzgerald et al., 2012; Yong et al., 2012) gained increasing attention as they successfully proved their value in efficiency improvement from ‘‘biological” side. Among these achievements, environmental factor (pH, metal ions, etc.) manipulation was considered to be one of the most promising approaches due to low cost and ease to operation. Electrolyte pH is an important parameter that affects the performance of MFCs. It was found that some EAB could take advantages of alkaline electrolyte (Yong et al., 2013; Yuan et al., 2011), while others preferred to acidic circumstance (Nimje et al., 2011). Thus, optimization of electrolyte pH is necessary and could significantly improve the performance of MFCs (Zhuang et al., 2010). Metal ions such as Fe3+, Ca2+, and Na+ were also explored to stimulate energy output of MFCs. Addition of Fe3+ (10 mM) increased the maximum power output of MFCs by 1.1 times (Wu et al., 2013), while Ca2+ addition (1.4 mM) enhanced the current output of MFCs by 80% (Fitzgerald et al., 2012). In addition, Na+ (20 g L 1) improved the maximum power output of MFCs by 30% (Lefebvre et al., 2012). These results suggested that pH optimization and metal ion addition would be effective ways to improve MFCs performance. However, adjust pH of bulk electrolyte is still cost intensive, and sometimes may be unavailable. Addition of metal ions would be more feasible in practical application. Heavy metal ions usually showed sophisticated effects on bacteria depends on their concentration, e.g., they are usually detrimental to bacteria at high concentration, while some of them might induce/stimulate the cellular metabolism at low concentration (Paraszkiewicz et al., 2007; Xu et al., 2014). However, the effects of heavy metal ions on EET and MFCs performance are still unclear. In this study, the effects of heavy metal ions on energy output of MFCs were investigated. Interestingly, it was found that nanomolar Cu2+ or Cd2+ significantly improved power output of MFCs by increasing electron shuttle accumulation and biofilm formation. The results provided valuable knowledge for MFCs operation and suggested trace heavy metal ion addition would be a practical approach for efficient EET and enhancement on energy output of MFCs.
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2.2. Bacteria and growth condition Shewanella oneidensis MR-1 was cultured at 30 °C in LB broth with shaking at 200 rpm until the optical density at 600 nm (OD600) reached about 5.0. The cells were collected by centrifugation (5000 rpm, 5 min) and washed with M9 for three times. Then, the cell pellets were re-suspended with electrolyte (95% M9 medium and 5% LB broth, 18 mM lactate) and were adjusted to the desired cell concentration (OD600 = 2.5), and were inoculated into the anodic chamber of the MFCs (30 ml). The effects of metal ions on the MFCs performance were tested by changing the metal ions’ final concentration. Each test was conducted in batch mode. Metal ions used in this study are copper (II) (CuSO4), cadmium (II) (CdCl2), zinc (II) (ZnCl2), chromium (III) (CrCl3), manganese (II) (MnCl2), cobalt (II) (CoCl2), nickel (II) (NiSO4). Stock solution (1 g L 1) of each metal ion was prepared with electrolyte and stored at 4 °C until use. For addition of metal ions, 300 lL of diluted (the times of dilution was dependent on the final concentration needed for MFCs) metal ion solution was immediately injected into the anodic chamber of MFCs after bacteria inoculation. Each metal ion and concentration is tested independently in batch mode, and all experiments are conducted in triplicates. 2.3. HPLC analysis of riboflavin The concentration of riboflavin was analyzed by using reverse phase high performance liquid chromatography (HPLC) according to the reported method (Yong et al., 2013). In brief, 2 ml of anodic culture suspension from MFCs was centrifuged (5000 rpm 5 min), and the supernatant was filtrated with 0.22 lm cellulose membrane and then subjected to HPLC analysis. HPLC analysis was performed at a Shimadzu HPLC system (Japan) equipped with a UV detector. The column used was a Shimadzu C18 analytical column (5 micro-m particle size, 250 mm 4.6 mm i.d.). Riboflavin was separated in a solvent system of water–methanol–acetic acid (68:32:0.1 v/v) and a flow rate of 1.0 mL min 1, the HPLC profile was monitored at 254 nm. 2.4. Quantitative biofilm detection
2. Materials and methods 2.1. MFCs set-up and electrochemical measurements Dual chamber MFCs (the size of each chamber being 3 cm (width) 5 cm (length) 6 cm (height)) separated with nafion 117 membrane (Dupont, USA) was used in this work (Yong et al., 2013, 2014b). Carbon cloth with size if 2 cm 3.5 cm and 1 cm 2 cm were used as cathode and anode electrodes, respectively. Saturated calomel electrode (SCE) electrode (CH Instrument, Shanghai, China) was inserted into each chamber as reference electrode when needed. The anodic medium was M9 medium supplemented with 18 mM sodium lactate as carbon source and electron donor (Yong et al., 2012, 2014b). The cathodic solution was 50 mM K3[Fe(CN)6] in 50 mM KCl solution. For current generation measurement, a 2 KX external resistor was connected into the circuit of MFCs, and the potential of the MFCs was recorded by a digital multimeter. Current density (I) was calculated as I = V (output voltage)/R (external resistance)/A (anode surface area), and power density (P) was calculated as P = V I. All other electrochemical measurements were conducted using CHI 660E electrochemical working station (CH Instrument, Shanghai, China). Power output and polarization curves were obtained by varying the external loading resistor (Qiao et al., 2008). The bacteria attachment on the anodes was observed with scanning electron microscopy (SEM) (JEOL, JSM-7001F, Japan).
Quantitative biofilm formation ability assay was conducted based on a crystal violet staining method (Morales-Calderon et al., 2012) with slight modification. Briefly, strain S. oneidensis MR-1 was cultured with shaking (200 rpm) in LB medium for 15 h at 30 °C. Then, 1 ml of culture suspension was centrifuged (5000 rpm 5 min). Then the cell pellets were re-suspended with 2 ml fresh medium (95% M9 medium and 5% LB broth, 18 mM sodium lactate). After that, 200 lL of cell suspensions were added into each well of 96-well plate. The plate was incubated at 30 °C for another 48 h. After that, planktonic cells were removed. The surface-associated biomass was stained by 0.5% crystal violet for 15 min. Then, the stained biofilm was dissolved with 200 lL of 95% ethanol, and the amount of biofilm biomass was quantified by A595 with a microplate reader (Tecan, Männedorf, Switzerland). 3. Results and discussion 3.1. Effect of metal ions on performance of MFCs Metal ions, in particular heavy metal ions, have marked impacts on the activity of microbes, which will affect the performance of MFCs as expected. The voltage outputs of MFCs with the addition of different metal ions were measured. As listed in Table 1, all the metal ions tested except Cd2+ exhibited inhibition on the voltage outputs of MFCs at certain concentration, which might due to
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Table 1 Effect of different metal ions on the voltage output of MFCs* and the minimum inhibitory concentration (MIC) for Cu2+ or Cd2+. Concentration 1 lg L 100 lg L 1 1 mg L 1 10 mg L 1 MIC (mg L 1) 1
Cu2+
Cd2+
Zn2+
Cr3+
Mn2+
**
+ + + + 300
+ +
+ +
+ + +
+ +
800
***
NT
NT NT
NT
Co2+
Ni2+
NT NT NT NT
NT NT NT NT
*
Each condition was tested independently at batch mode. + Indicates enhancement; + indicates no significant effect; repression. *** NT, not tested. **
indicates
their toxic effect on S. oneidensis MR-1. For example, Co2+ and Ni2+ showed significant inhibition at the concentration of 1 lg L 1. As these heavy metal ions are toxic to bacteria, it is normal to expect these inhibition phenomena. Surprisingly, Cu2+ and Cd2+ enhanced the voltage output of MFCs significantly, once the concentration is lower than 100 lg L 1 and 1 mg L 1, respectively (Table 1). This unexpected exploration is interesting as it might be used to improve the MFCs performance. Thus, the effects of Cu2+ and Cd2+ on MFCs performance were systematically investigated. To get the highest energy output of MFCs, the addition concentrations of these two heavy metal ions were optimized. As shown in Fig. 1, the maximum voltage outputs of MFCs with the addition of Cu2+ or Cd2+ at different concentration were monitored. Compared to the control (without addition) with the maximum voltage output of 117 ± 5 mV, supplemented with Cu2+ and Cd2+ showed great enhancement of bioelectricity generation under the concentration lower than 100 lg L 1. In the assay supplemented with Cu2+, the highest voltage output of 165 ± 6 mV was achieved at the concentration of 1 lg L 1, which was 1.4 times of the control. Similarly, for Cd2+, it was found that the voltage output was enhanced when the concentration is lower than 1 mg L 1. The highest voltage output of 175 ± 8 mV was obtained with the addition of 1 lg L 1 Cd2+, which was 1.5 times of the control. Previous research showed that Fe3+ (6 mM) supplementation in S. oneidensis MR-1 inoculated MFCs increased the electricity generation (Wu et al., 2013). In addition, Fitzgerald et al. found that addition of CaCl2 (>300 lM) increased the current density by >80% in S. oneidensis MR-1 inoculated MFCs (Fitzgerald et al., 2012). These findings suggested that addition of metal ions in MFCs was an effective way for performance improvement. However, addition of heavy metal ions in MFCs is seldom studied due to high concentration of heavy metal ions are detrimental to cells and might result in water pollution. Interestingly, this study found that heavy metal ions (Cu2+ or Cd2+) at the concentrations 105 times lower than their the minimum inhibitory concentrations (MIC) also significantly improved MFCs performance. Thus, the finding of this study highlighted that heavy metal ions addition would also be another promising and practical for improvement of MFCs performance.
Fig. 1. Effect of Cu2+ or Cd2+ on the maximum voltage output of MFCs. Each condition was tested independently at batch mode. Three replicates are performed for each experiment.
3.2. Enhancement of MFCs performance by Cu2+ and Cd2+ addition Then, the effect of Cu2+ or Cd2+ on MFCs performance was investigated in details, respectively. As shown in Fig. 2a, the voltage output of MFCs steadily increased after bacteria inoculation and reached its maximum value of 117 mV for MFCs without heavy metal addition at the first cycle. For MFCs supplemented with Cu2+ or Cd2+, the voltage outputs increased faster than that of the control, and higher maximum values were obtained (about 169 ± 6 mV for Cu2+ addition, and about 175 ± 8 mV for Cd2+ addition). At the following cycles, the reproducible voltage output was obtained by feeding fresh sodium lactate as electron donor. The
Fig. 2. Performance of MFCs supplemented with different metal ions. (a) Timecourse profiles of voltage outputs (MFC with a 2 KX exterior resistor) of MFCs with (1 lg L 1 Cu2+ or Cd2+) or without (control) metal ions addition. Arrows indicates lactate (final concentration, 18 mM) was added. (b) Power output and polarization curves of MFCs with (1 lg L 1 Cu2+ or Cd2+) or without (control) metal ions addition.
polarization and power output curves, which were obtained by varying external loading resistance, were plotted to investigate the effect of Cu2+ or Cd2+ on the power output of MFCs (Fig. 2b). The maximum power density of the MFCs supplemented with Cu2+ was 92.4 mW m 2, which was 1.3 times higher than that of
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the control (39.8 mW m 2). Similarity, The maximum power density of the MFCs supplemented with Cd2+ was 104.4 mW m 2, which was 1.6 times higher than that of the control. Meanwhile, it was found that the open circuit voltage (OCV) of the MFCs supplemented with Cu2+ (676 mV) or Cd2+ (673 mV) was higher than that of the control (579 mV), which might favor EET and power output. These results confirmed that trace amount of Cu2+ and Cd2+ could improve the energy output of S. oneidensis MR-1 inoculated MFCs. Some metal ions may serve as trace element for bacteria to improve cell growth at low concentration, which might in turn enhance MFCs performance. Thus, the effect of Cu2+ or Cd2+ on cell growth of S. oneidensis MR-1 was further investigated. At high concentration (10 mg L 1), both of Cu2+ and Cd2+ showed slight growth inhibition (Fig. S1). However, according to the cell growth curves, there was no significant difference between the control and that with trace level (1 lg L 1, 105 times lower than MIC) of Cu2+ or Cd2+ addition. The results excluded the possibility of cell growth promotion related MFCs performance improvement by Cu2+ or Cd2+, and suggested that the power output improvement by Cu2+ or Cd2+ addition should be ascribed to other factors besides cell growth. 3.3. Increased riboflavin production by Cu2+ and Cd2+ addition Extracellular electron transfer (EET) efficiency was considered to be the limiting parameter that determined the performance of MFCs (Yang et al., 2012). To date, there are two EET pathways have been identified for EAB, i.e., direct electron transfer through outer membrane redox active proteins (e.g., c-type cytochromes) or conductive nanowire, and indirect electron transfer mediated by exogenous or endogenous electron shuttles (e.g., pyocyanin, riboflavin) (Lovley, 2006; Yang et al., 2012). S. oneidensis MR-1 usually employed c-type cytochrome for direct EET and indirect EET mediated by electron shuttle of riboflavin (Marsili et al., 2008; Yong et al., 2014b). To test the effect of metal ions on EET of S. oneidensis MR-1 in MFCs, cyclic voltammetry (CV) was conducted when the voltage outputs reached the steady state. As illustrated in Fig. 3a, MFCs with or without metal ions showed clear catalytic wave with mid-point potential 0.42 V (vs SCE), which was in agreement with the electrochemical reaction of riboflavin (the main electron shuttle of S. oneidensis MR-1) (Yong et al., 2013). Interestingly, the catalytic currents of CV obtained from MFCs with heavy metal ions addition (regardless of Cu2+ or Cd2+) (j2 shown in Fig. 3a) were much higher than that of the control (j1 shown in Fig. 3a). The results substantiated that trace level of heavy metal ions improved electron shuttle mediated EET, which might contribute to the enhancement of MFCs power output. It also implied that heavy metal ions might induce overproduction of riboflavin in S. oneidensis MR-1. The concentration of riboflavin in MFCs was further analyzed by HPLC after MFCs discharge. As shown in Fig. 3b, riboflavin concentrations detected in the MFCs with Cu2+, or Cd2+ are 0.67 ± 0.02, and 0.65 ± 0.01 lg mL 1, respectively, which were 21.8%, and 18.2% higher than that of the control (0.55 ± 0.01 lg mL 1). The result confirmed the CV analysis and indicated that heavy metal ions improved riboflavin production of S. oneidensis MR-1. Heavy metal ions including Cu2+ and Cd2+ are usually used as inducers to improve the production of secondary metabolites by plants, fungi or bacteria (Maksymiec et al., 2005; Morgenstern et al., 2015; Xu et al., 2014). For example, Cu2+ or Cd2+ addition induced the accumulation of jasmonates in mature leaves of Arabidopsis thaliana (Maksymiec et al., 2005). Co2+ was also proved to be an effective inducer on antibiotic production by Streptomyces coelicolor (Morgenstern et al., 2015). Here, this study demonstrated that Cu2+ or Cd2+ at trace level could induce enhancement on
Fig. 3. (a) Cyclic voltammograms (CV) of MFCs with (1 lg L 1 Cu2+ or Cd2+) or without (control) metal ions addition. ‘‘j1” and ‘‘j2” indicates the catalytic current of the corresponding CV profiles, respectively. (b) Riboflavin concentration in MFCs with (1 lg L 1 Cu2+ or Cd2+) or without (control) metal ions addition analyzed by HPLC. Three replicates are performed for each experiment.
riboflavin (a typical secondary metabolite of bacteria) production (Fig. 3b). As riboflavin is the major endogenous electron shuttle of S. oneidensis MR-1 that mediated the indirect EET between bacteria and electrode, the improved riboflavin production underlies the enhancement of energy output by heavy metal ions. 3.4. Enhanced anodic bacterial attachment by Cu2+ and Cd2+ addition Anodic biofilm, as a bacterial community adhered to the anode of MFC, also played important roles for power generation in MFCs. Better anodic biofilm formation usually followed with higher power output (Yu et al., 2011). Increase of biofilm formation is a common protective mechanism for bacteria when exposed to heavy metal ions, as biofilm can withstand higher toxicity (Morales-Calderon et al., 2012). The sub-lethal level of toxic chemicals may induce extensive synthesis of extracellular polysaccharide and enhancement on c-di-GMP signaling, which in turn improve the biofilm formation (Chua et al., 2015). Thus, the formation of anodic biofilm under different conditions was observed by SEM after MFCs discharge. As shown in Fig. S2, there are only a few bacteria attached on the surface of the anodic electrode due to poor biofilm formation ability of S. oneidensis MR-1. However, after Cu2+ addition, much more cells attached on the surface of the electrode were observed. The increased bacteria attachment was also observed after Cd2+ addition.
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51578266, 21306069), Natural Science Foundation of Jiangsu Province (BK20130492), Specialized Research Fund for the Doctoral Program of Higher Education (Ministry of Education, 20133227120014).
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.biortech.2016.03. 144. References
Fig. 4. Quantitative analysis of biofilm formation ability of S. oneidensis MR-1 (control, 1 lg L 1 Cu2+ and 1 lg L 1 Cd2+). At least five replicates are performed for each experiment.
To further confirm the SEM observation, another biofilm formation assay was conducted. In this assay system, the biofilm biomass formed on the wall of 96 micro-wells were stained and quantified. As shown in Fig. 4, the biofilm formation ability of S. oneidensis MR-1 was significantly enhanced by Cu2+ or Cd2+ addition. Compared with the control without any addition, the biofilm formation of S. oneidensis MR-1 was increased about 45% with Cu2+ addition, while it increased about 29% with Cd2+ addition. Although the electrodes (carbon fiber) and 96 micro-wells (polypropylene) are made from different material, Cu2+ or Cd2+ addition could increase the bacteria attachment on the surface of both materials. The results suggested Cu2+ and Cd2+ might induce a general pathway to enhance the bacterial attachment on abiotic surface. The results implied that enhancement of bacteria attachment on the anodic electrode by heavy metal ions might also contribute to the power output improvement. It is interesting that there are much more cells attached on the electrode surface with Cu2+ addition than that with Cd2+ addition (Fig. S2), while the power output of MFCs under these two different conditions was nearly the same (Fig. 2). The results implied that enhanced anodic bacteria attachment and electron shuttle concentration might just partially contributed to the power output improvement, other factors related to power output such as cellular metabolism and activity of electron transfer chain (e.g., c-type cytochrome) might be affected by Cu2+ or Cd2+ and might play a role for energy output improvement. 4. Conclusion This study showed an interesting concentration-dependent effects of heavy metal ions on the performance of MFCs. Impressively, Cu2+ and Cd2+ at trace level (5–6 nM) significantly improved MFCs performance, i.e., 1.3–1.6 times higher power output was obtained compared to that of the control. Further analyses indicated that enhancement on electron shuttle synthesis (riboflavin) and anodic biofilm formation was the underlying mechanism for heavy metal ions improved EET as well as MFCs performance. These results provided mechanistic insights into the effect of heavy metal ions on the performance of MFCs, which is valuable for practical operation of MFCs. Acknowledgements The authors sincerely acknowledge the financial supports from National Natural Science Foundation of China (NSFC grant Nos.
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