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Unveiling characteristics of dye-bearing microbial fuel cells for energy and materials recycling: Redox mediators
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journal homepage: www.elsevier.com/locate/he
Unveiling characteristics of dye-bearing microbial fuel cells for energy and materials recycling: Redox mediators Bor-Yann Chen a,*, Chung-Chuan Hsueh a, Shi-Qi Liu a, Jhao Yin Hung a, Yan Qiao b, Pei-Lin Yueh a, Yu-Min Wang a a b
Department of Chemical and Materials Engineering, National I-Lan University, I-Lan 26047, Taiwan Institute for Clean Energy & Advanced Materials, Southwest University, Chongqing 400715, China
article info
abstract
Article history:
This study disclosed why and how some decolorized intermediates (e.g., 2-aminophenol)
Received 26 December 2012
could act as electron-shuttling mediator(s) to enhance the capabilities of reductive decol-
Received in revised form
orization and bioelectricity generation. It also selected several model auxochrome-
17 March 2013
containing compounds structurally associated to 2AP to explore how chemical structure
Accepted 25 March 2013
influenced the feasibility of possible electron shuttles for power producing capabilities in
Available online 21 April 2013
microbial fuel cells (MFCs). The selection criteria of electron-shuttling mediators were suggested for optimal reductive decolorization and bioelectricity generation in MFCs for
Keywords:
practical application.
Auxochrome
Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Electron shuttle
reserved.
Microbial fuel cell Reductive decolorization
1.
Introduction
As an energy-famished country e Taiwan, renewable energy is always being considered as a promising alternative to help reduce the reliance on fossil fuels. In fact, due to Taiwan’s abundant bioresources significant attentions of biomassbased energy (BBE) have been paid nowadays as one of priority resources of renewable energy, in particular after the unfolding crisis with Japan’s devastating earthquake, tsunami and crippled unclear reactor meltdown in March, 2011. Among all BBE sources, microbial fuel cells (MFCs) can use naturallyoccurring microbes as biocatalysts to extract biofuel energy from oxidation of organic matter in wastewater for sustainable development. Recently, Lay et al. [1] used starchcontaining wastewater collected from Taiwan’s textile industry as a potential bioenergy source for biohydrogen
production. In addition, MFCs could simultaneously expedite bioelectricity generation as well as pollutant degradation during wastewater treatment [2,3]. In fact, electrochemical activities of microorganisms [4] significantly influenced the performance of bioelectricity production in MFC through at least three mechanisms: electron shuttling cell-secreting mediators (e.g., phenazine, quinones), membrane-bound redox proteins (e.g., mobile electron carriers such as cytochromes), and conductive pili (or nanowires) (e.g., wired communities of Geobacter sulfurreducens, Shewanella oneidensis) [5]. Regarding dye-bearing wastewater treatment, Cao et al. [6] and Li et al. [7] presented pioneer works on simultaneous dye decolorization (ca. <5% not decolorized) and power generation (ca. max. power density w500e600 mW m2) using mixed consortia-bearing MFC systems. Recently, Chen et al. [8] uncovered microbial characteristics of simultaneous
* Corresponding author. Fax: þ886 39357025. E-mail addresses: [email protected], [email protected] (B.-Y. Chen). 0360-3199/$ e see front matter Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijhydene.2013.03.132
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 1 5 5 9 8 e1 5 6 0 5
bioelectricity generation and dye decolorization (SBG&DD) using pure biodecolorizers (e.g., Klebsiella pneumoniae, Proteus hauseri), suggesting that reductive decolorization and bioelectricity generation of biodecolorizers was competitive to each other. Moreover, Chen et al. [9] quantitatively revealed that electrons extracted from this electron-transport chain of bacterial decolorizer via respiratory complexes were provided for either bioelectricity generation or color removal. They also successfully used 2-aminophenol (2AP) as a model exogenous mediator to enhance the performance of SBG&DD of P. hauseri. However, the mysteries why some amine phenolic compounds (e.g., 2AP) could be used to skyrocket the efficiency of SBG&DD were still remained uncertain to be explored. To decipher the mysteries of electron-shuttling characteristics of amine phenolic compounds augmentation, this study conducted cyclic voltammetric (CV) inspections upon compounds with similar functional groups. Comparison upon CV profiles of these possible mediators clearly suggested why and how they could or could not play as a role of electron shuttle. This comparative assessment can provide selection criteria of candidate electron transfer mediator for optimal power generation in MFC. These operation guidelines could provide professionals promising criteria to examine the feasibility of SBG&DD in MFCs before they launch practically.
2.
Methods
2.1.
Electrochemical measurements
Cyclic voltammetry of amine phenolic compounds was performed using an electrochemical workstation (Jiehan 5600, Taiwan) at 10 mV s1 scan rate. The working, counter, and reference electrodes were, respectively, a glassy carbon electrode (0.07 cm2), platinum electrode (6.08 cm2), and a Hg/Hg2Cl2 electrode filled with saturated KCl(aq). The glassy carbon electrode (GCE, ID ¼ 3 mm; model CHI104, CH Instruments Inc., USA) was successively polished with 0.05 mm alumina polish and then rinsed with 0.5 M H2SO4 and deionized water before use. The experiments were performed in phosphate buffer solutions (pH ¼ 7.0) at 0.1 M and the solutions were purged with nitrogen for 15 min prior to analysis. The scanning rate was 10 mV s1 over the range from 0.4 to 0.6 V [5]. The redox potentials recorded as Hg/Hg2Cl2 reference electrode were corrected by 0.241 V (i.e., E0 of Hg/Hg2Cl2) to the standard hydrogen electrode (SHE).
2.2.
MFC construction and microbial cultures
Membrane-free air cathode single-chamber MFCs (refer to Ref. [10] for schematic setup of MFC) were constructed in cylindrical tubes made by polymethyl methacrylate (PMMA) (cell sizing ID ¼ 54 mm, L ¼ 95 mm) with the operating volume of ca. 220 mL. Porous carbon cloth (CeTech) (without waterproofing or catalyst) with a projected area of ca. 22.9 cm2 (i.e., p 2.72) on one side was used as anode electrodes. The air cathode was almost identical to the anode in size and consisted of a polytetrafluoroethylene (PTFE) diffusion layer (CeTech) on the air-facing side.
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The procedures of bacterial cultures for MFCs were described elsewhere [11]. Culture medium in MFCs used in the study (unit: g L1) is 0.2 LB (tryptone 2, yeast extract 1, sodium chloride 10). A loopful of bacterial seed taken from an isolated colony on a LB-streak plate (i.e., LB medium supplemented with Bacto agar 20 g L1) was precultured in 50 mL LB broth laden with 200 mg L1 reactive blue 160 (RBu160; CAS 71827-76-9) using 250 mL Erlenmeyer flask for 12 h overnight at 30 C, 125 rpm. Cultured cells were then collected after centrifugation at 2400 g for 10 min. After cell-free supernatant was discarded, the biomass was rinsed and mixed with DI/DD (deionized/distilled) water. Rinse-and-mix procedures were repeated twice to harvest residue-free 2 rinsed biomass and then 2 rinsed biomass was concentrated with DI/DD water in 5 mL for inoculation to MFC.
2.3.
Experimental operations
Acclimation step: To uncover the effect of amine phenolic compounds (B12d, 12db) for stimulating bioelectricity generation and dye decolorization, approximately 5 mL concentrated biomass was well-mixed with 2.5 mL sterilized 0.2 LB medium in MFCs for acclimatization. During serial acclimation, every 48 h approximately 5 mL cell broth was meticulously replaced by impulse injection of 5 mL fresh sterile 4.4 LB medium to maintain initial concentration at 0.2 LB. Note that “impulse injection” indicated that a fresh nutrient medium was supplemented immediately in a very short period of time [12]. Then, the steady-state output power generation of MFCs was approximately achieved after approx. 30 days acclimation [9]. Experimental step: To explore stimulating characteristics of B12d for bioelectricity generation, the batch-fed mode of MFC operation with impulse injection of energy substrate was carried out at 25 C every 100 h. That is, 5.0 mL of 8.8 LB broth laden with appropriate concentrations of amine phenolic compound 12db or B12d was supplemented to MFCs to maintain culture medium in 0.2 LB for inspection. Approx. 1 h after impulse injection of energy substrate, the supplemented medium was considered to be well-distributed in MFC and then electrochemical analysis of MFCs was conducted.
2.4.
Sudan black B staining
Several drops (ca. 10 mL) of 12e16 h LB-culture cell broth (selected from cultured bacteria in different MFCs) were fixed on a glass slide by applying heat and then stained with a 3% Sudan black B (SB) (w/v in 70% ethanol, Sigma) solution for 10 min. The slide was then immersed in xylene until completely decolorized. The sample was counterstained with safranin (5%, w/v, in distilled water, Sigma Inc.) for 10 s, twice washed with distilled water, and dried afterward. Once the slide was completely dried, several drops of immersion oil were directly added on cell sample for microscopic examination under optical microscopy (Leica, Tokyo, Japan) [13]. Cellular compartments shown in black blue and pink color under optical microscopic observation indicated possible PHA-containing and PHA-absent granules in cell samples, respectively.
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3.
Results and discussion
3.1.
Assessment upon electron-shuttling mediators
Prior study [12] mentioned that bioelectricity generation and reductive decolorization were both competitive to each other. To uncover the mysteries behind bioelectricity generation and dye decolorization, the capability of amine phenolic compound (e.g., isomeric aminophenols; 2, 3, or 4-AP) [14] to enhance reductive decolorization was quantitatively disclosed. In fact, electron-shuttles (i.e., redox mediators) were reported to have capabilities to be reversibly oxidized and reduced as an electron carrier in multiple redox reactions. As a matter of fact, prior studies [15,16] also confirmed that 2-aminophenol (2AP) and benezene-1,2-diol (B12d) were electron-shuttling mediators to enhance bioelectricity generation and reductive decolorization based upon various MFC data. However, why and how some aminophenol isomers were capable to be electron-shuttling mediators was still remained open for discussion. To disclose such mysteries of electron-mediating properties, cyclic voltammetric inspections upon several compounds with similar functional groups (1,2-, 1,4-diaminobenezene (12db, 14db), 3-,4aminobenzoic acid (3ABA, 4ABA), aniline-2-sulfonic acid (A2SA), aniline-4-sulfonic acid (A4SA)) were carried out. As shown in Fig. 1, evidently only 2AP and 4AP could show capabilities of quasi-reversibility of redox processes for a separation of peak potentials of electron-shuttling mediators [15]. Regarding exogenous electron-shuttling mediator, prior study [8] suggested that phenyl methadiamine produced via reductive decolorization could mediate electron transfer in anodic biofilm of P. hauseri for power generation in MFC [10]. As indicated, appropriate amount of 2AP supplemented could significantly enhance the performance of bioelectricity generation in MFC. The output voltages were gradually increased with respect to an increased concentration of 2AP, suggesting that current production of P. hauseri in MFC could be significantly stimulated by the supplementation of 2AP as an exogenous electron-transfer mediator. However, not all isomeric aminophenols could effectively work as redox mediators or electron shuttles for extracellular electron transfer from P. hauseri cells to the anode (e.g., the supplementation of 3AP). As shown in Fig. 1, the absence of reduction and oxidation peak potentials for 3AP in its CV profile led to low feasibility for 3AP to be an electron-shuttling mediator. Similar example of 1,3dihydroxybenzene (or resorcinol) [17] also showed that meta-isomer was not appropriate to be an electron shuttle. Wang et al. [17] mentioned that “the electro-oxidation reaction of resorcinol (or 1,3-dihydroxybenzene) (a meta-isomer) shows an irreversible process, because no corresponding reductive peak was observed on the cathodic branch.” In addition, cyclic voltammogram for the redox processes of 1 mM benzene-1,3-diol could not show the presence of the oxidation or reduction potential (data not shown). Moreover, although 4AP could show the presence of reduction and oxidation peak potentials in CV profile (Fig. 1), zero output voltage was still observed in 4AP-supplemented MFC due to significant toxicity of 4AP on P. hauseri as shown elsewhere [8,10]. This point also confirmed that a reduced dye (i.e., aromatic amine) with relatively less biotoxicity might be feasible
as an exogenous mediator to stimulate electron transfer to anodic biofilm in MFC (e.g., 2AP). In fact, 2AP was also found to be a possible electron-shuttling mediator to Gram-negative aerobic rod Acinetobacter sp. 72 and facultatively anaerobic Gram-negative rod Enterobacter sp. m30 seeded mixed culture MFC. However, the mysteries why 2AP could play as an electron-shuttling mediator was still needed to be disclosed for optimization of power generation in MFC.
3.2.
Electron-mediating mechanism
In fact, the reversible redox properties of 2AP and 4AP were due to the reasons as follows: The electron shuttle can be reversibly interchanged between its reduced and oxidized form for mediating electron-transfer processes involved in energy production. For example, the inter-conversion of hydroquinone (p-dihydroxybenzene; a reduced form) and quinone (cyclohexa-2,5-diene-1,4-dione; an oxidized form) through the formation of active radicals (e.g., RD2 and resonance forms RD1A, RD1B in Fig. 2) can be reversibly reoxidized and re-reduced (Fig. 2 as illustrated in Ref. [18]). When hydroxyl groups are present on the benzene ring of the compounds (e.g., 2AP, 4AP, hydroquinone), these molecules can express significant electron-shuttling capabilities for electricity generation. As a matter of fact, this was the reasoning why chemicals 14db, A2SA, A4SA, 4ABA, and 3ABA (except of 12db which was lack of hydroxyl groups) could not own such shuttling functions as electron-transport mediators in MFC. However, 12db could play as an electron shuttle to enhance bioelectricity generation, since 12db could be oxidized via dimerization to be 1,2-diaminophenazine (Fig. 4(A)) [19]. As a matter of fact, 1,2-diaminophenazine could also play as redox mediator (Fig. 4(B); [16,17]). In addition, due to acidic characteristics of hydroxyl groups (eOH) of phenols, the acidic hydrogen can be dissociated to obtain phenolate anions, and then phenolate anions will be favorably oxidized to form active radicals (e.g., RD1A, RD1B, and RD2 in Fig. 2). Apparently, these steps of free-radical formation are crucial steps to generate redox mediators to mediate electron-transfer processes for energy and/or electricity production. For example, many derivatives of naphthoquinones or anthraquinones with the free-radical forming characteristics were also capable to be redox mediators [20,21]. In contrast, other functional groups (e.g., carboxyl (eCOOH) and sulfo (eSO3H) group) do not have such capabilities to play as a role of redox mediator. Although they owned an acidic hydrogen (Hþ) to be dissociated as an anion, they could not be easily oxidized to form free radicals afterward (e.g., RD1 in Fig. 2, RD3A, RD3B RD4A, and RD4B in Fig. 3(A, B)). Moreover, as hydrogen atom on the amino group (eNH2) of 14db is not acidic, it is thus not thermodynamically favorable to be deprotonated to form an anion. Thus, the compounds with amino group are not viable to be oxidized to form radical intermediates on the benzene ring (e.g., hydroquinone anion in Fig. 2) for electron transfer. That is, the compounds without hydroxy (eOH) group will not express electron-shuttling characteristics. Moreover, although all three AP isomers (i.e., o-, m-, p-isomers) contained hydroxyl groups, only 2 isomeric compounds
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Fig. 1 e CV profiles of model anxochrome-containing compounds in the study (N.D. denoted not determined).
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Fig. 2 e Proposed pathways of inter-conversion between hydroquinone and quinone.
(A)
(B)
(C)
Fig. 3 e (A) Proposed pathways of inter-conversion between 2AP and o-quinonimine, (B) Proposed pathways of interconversion between 4AP and p-quinonimine, (C) Proposed pathways of inter-conversion between 3AP and intermediates CI1-3.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 1 5 5 9 8 e1 5 6 0 5
(A)
(B)
Fig. 4 e (A) Enzymatic dimerization of 1,2-diaminobenzene (12 db) to form electron shuttles. (B) Electron shuttling route for 1,2-diaminobenzene (12 db).
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2AP (o-isomer) and 4AP (p-isomer) could own electronshuttling capabilities. Similar to hydroquinone, 2AP and 4AP can be favorably oxidized to form o- and p-quinoniminium ion (i.e., iminium ion (¼NHþ2) containing chemical), and then be deprotonated to form imine (¼NH) containing o- and p-quinonimine structurally similar to electron shuttles 1,2benzoquinone (ortho-quinone) and 1,4-benzoquinone ( paraquinone), respectively, as shown in Fig. 3. However, due to characteristics of meta-isomer, 3AP cannot carry out such inter-conversion of reduced and oxidized form for electron-shuttling capabilities to mediate energy transfer in MFC. Since 3AP cannot carry a positive charge on benzene ring after oxidation, the positive charge will not be located on the carbon bonding to amino group as indicated in 2AP and 4AP (Fig. 3(A, B)). Thus, the positive charge at ortho or para position to the carbon that binds to amino group in oxidized 3AP (e.g., cationic intermediates CI1, CI2, CI3, CI4, and CI5 in Fig. 3(C)) will not be capable to form iminium ion. Similar to
Fig. 5 e (A) Optical microscopic observation upon possible PHA accumulation of bacterial cells of mixed consortia obtained from Aeromonas sp. C78, Aeromonas hydrophila NIU01, Acinetobacter johnsonii NIUx72, Proteus hauseri ZMd44, Enterobacter cancerogenus BYm30-seeded microbial fuel cells after Sudan-black staining (black colors (i.e., black-circled portions) and purple colors (i.e., red-circled portions) denoted (D) and (L) response of possible strains, respectively). (B) Comparison of dry cell weight, PHB concentration and PHB content of samples taken from Enterobacter cancerogenus BYm30, Proteus hauseri ZMd44, Acinetobacter johnsonii NIUx72, Aeromonas hydrophila NIU01 and Aeromonas sp. C78 seeded MFCs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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300
250
6th
15th 200
BYm30-M ZMd44-p NIUx72-M NIUx73-2 ZMd30-2 ZMd31-3 NIU01-M
200 150 100 50 0 0.0
[RBu160] (mg L-1)
[RBu160] (mg L-1)
250
BYm30-M ZMd44-p NIUx72-M NIUx73-2 ZMd30-2 ZMd31-3 NIU01-M
150
100
50
0 0.5
1.0
1.5
2.0
2.5
0.0
Time (hr)
0.5
1.0
1.5
2.0
2.5
Time (hr)
Fig. 6 e Time-series profiles of dye decolorization of different bacterial strain-seeded MFCs after 6th and 15th impulse injection of reactive blue 160 (RBu160). Strains BYm30, ZMd44, NIUx72, NIUx73, ZMd30, ZMd31 and NIU01 denoted Enterobacter cancerogenus, Proteus hauseri, Acinetobacter johnsonii, Aeromonas hydrophila, Klebsiella pneumoniae, Klebsiella pneumoniae and Aeromonas hydrophila, respectively.
aforementioned reasoning for 3-AP, 1,3-dihydroxybenzene could thus not be feasible as hydroquinone-like redox mediator [17]. Apparently, further deprotonation of oxidized 3AP to obtain imine as shown in 2AP and 4AP was not thermodynamically favorable. That is to say, 3AP can not play as a role of electron-shuttling mediator. These analyses uncovered the mysteries why and how only 2AP and 4AP can be electronshuttling mediators for energy recycling in MFC as mentioned herein.
3.3. MFCs
PHB accumulation and wastewater decolorization in
As aforementioned, mediator supplementation to microbial fuel cells could enhance energy-extraction through oxidation of organic matter in wastewater for energy recycling. As a matter of fact, MFC can also produce biopolymer during wastewater decolorization for simultaneous materials recycling. Here, optical microscopic observations upon bacterial cells of mixed cultures of Aeromonas sp. C78, Aeromonas hydrophila NIU01, Acinetobacter johnsonii NIUx72, P. hauseri ZMd44, Enterobacter cancerogenus BYm30 seeded mixed culture MFCs after SB staining indicated that significant black blue granules accumulated in these microbial cells in MFCs (Fig. 5). To uncover the profiles of PHA accumulation in MFCs, samples of these MFCs were taken for determining intracellular content of polyhydroxybutyrate (PHB) as an indicator
Table 1 e Tabulated list of initial decolorization rate (IDR) of various bacterium-seeded MFCs after 6th and 15th impulse injection of RBu160 (unit: mg LL1 hL1). MFC
IDR6
IDR15
BYm30-M ZMd44-p NIUx72-M NIUx73-2 ZMd30-2 ZMd31-3 NIU01-M
381.07 873.70 234.76 414.46 753.88 727.56 471.42
673.46 698.89 379.86 157.62 656.40 607.71 432.27
PHA. Fig. 6 indicated that only samples obtained from BYm30 and ZMd44-seeded MFCs contained significant amounts of PHB. This implied that those cultures were likely feasible candidate microorganisms for biodegradable polymer-polyhydroxyalkanoate (PHA) production. This finding showed that dye decolorization in parallel with bioelectricity generation and PHA production in MFC for energy and materials recycling was technically viable for sustainable development. Moreover, to reveal the feasibility of long-term stable simultaneous bioelectricity generation and reductive decolorization for MFC operation, dye decolorization profiles of several mixed culture-based MFCs were determined (Fig. 6, Table 1). As Table 1 indicated, after 6th and 15th impulse injection of reactive blue 160 (refer to Fig. 1 in Ref. [9] for operation procedure), color removal performance of MFCs still remained biologically stable. The points as aforementioned directly disclosed the promising feasibility of simultaneous energy and biomaterials recycling during wastewater decolorization.
4.
Conclusions
This study disclosed why and how some decolorized intermediates (e.g., 2-aminophenol) could act as electronshuttling mediator(s) to enhance the capabilities of reductive decolorization and bioelectricity generation. It also selected several model auxochrome-containing compounds structurally associated to 2AP to explore how chemical structure influenced the feasibility of possible electron shuttles for power producing capabilities in microbial fuel cells (MFCs). The selection criteria of electron-shuttling mediators were suggested for optimal reductive decolorization and bioelectricity generation in MFCs for practical application.
Acknowledgment The authors sincerely appreciate financial support (NSC 982221-E-197-007-MY3 and NSC 100-2621-M-197-001) from
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 1 5 5 9 8 e1 5 6 0 5
Taiwan’s National Science Council for the project of Microbial Fuel Cell (MFC)sdg conducted in Biochemical Engineering Lab, NIU.
references
[1] Lay CH, Kuo SY, Sen B, Chen CC, Chang JS, Lin CY. Fermentative biohydrogen production from starchcontaining textile wastewater. Int J Hydrogen Energy 2012;37:2050e7. [2] Du Z, Li H, Gu T. A state of the art review on microbial fuel cells: a promising technology for wastewater treatment and bioenergy. Biotechnol Adv 2007;25:464e82. [3] Rabaey K, Verstraete W. Microbial fuel cells: novel biotechnology for energy generation. Trends Biotechnol 2005;23:291e8. [4] Logan BE, Regan JM. Electricity-producing bacterial communities in microbial fuel cells. Trends Microbiol 2006;14:512e8. [5] Logan BE. Microbial fuel cell. New Jersey: John Wiley & Sons; 2008. [6] Cao Y, Hu Y, Sun J, Hou B. Explore various co-substrates for simultaneous electricity generation and congo red degradation in air-cathode single-chamber microbial fuel cell. Bioelectrochemistry 2010;79:71e6. [7] Li Z, Zhang X, Lin J, Han S, Lei L. Azo dye treatment with simultaneous electricity production in an anaerobiceaerobic sequential reactor and microbial fuel cell coupled system. Bioresour Technol 2010;101:4440e5. [8] Chen BY, Zhang MM, Chang CT, Ding Y, Lin KL, Chiou CS, et al. Assessment upon azo dye decolorization and bioelectricity generation by Proteus hauseri. Bioresour Technol 2010;101:4737e41. [9] Chen BY, Wang YM, Ng IS. Understanding interactive characteristics of bioelectricity generation and reductive decolorization using Proteus hauseri. Bioresour Technol 2011;102:1159e65.
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[10] Chen BY, Zhang MM, Ding Y, Chang CT. Feasibility study of simultaneous bioelectricity generation and dye decolorization using naturally occurring decolorizers. J Taiwan Inst Chem E 2010;41:682e8. [11] Hsueh CC, Chen BY. Exploring effects of chemical structure on azo dye decolorization characteristics by Pseudomonas luteola. J Hazard Mater 2008;154:703e10. [12] Chen BY, Wang YM, Ng IS, Liu SQ, Hung JY. Deciphering simultaneous bioelectricity generation and dye decolorization using Proteus hauseri. J Biosci Bioeng 2012;113:502e7. [13] Burdon KL. Fatty material in bacteria and fungi revealed by staining dried, fixed slide preparations. J Bacteriol 1946;52:665e78. [14] Vasconcellos LCG, Oliveira CP, Castellano EE, Ellena J, Moreira I´S. Structure and properties of ironecyclam complex of 2-aminophenol. Polyhedron 2001;20:493e9. [15] Peter W. Reduction potentials of one-electron couples involving free radicals in aqueous solution. J Phys Chem Ref Data 1989;18:1637e755. [16] Chen BY, Hsueh CC, Liu SQ, Ng IS, Wang YM. Deciphering mediating characteristics of decolorized intermediates for reductive decolorization and bioelectricity generation. Bioresour Technol 2013. http://dx.doi.org/10.1016/ j.biortech.2012.12.164. [17] Wang Z, Li S, Lu Q. Simultaneous determination of dihydroxybenzene isomers at single-wall carbon nanotube electrode. Sensors Actuators B 2007;127:420e5. [18] Carey FA, Sundberg RJ. Advanced organic chemistry part A: structure and mechanisms. 5th ed. Springer; 2007. [19] Tarcha PJ, Chu VP, Whittern D. 2,3-diaminophenazine is the product from the horseradish peroxidase-catalyzed oxidation of o-phenylenediamine. Anal Biochem 1987;165:230e3. [20] Van der Zee FP, Cervantes FJ. Impact and application of electron shuttles on the redox (bio)transformation of contaminants: a review. Biotechnol Adv 2009;27:256e77. [21] Watanabe K, Manefield M, Lee M, Kouzuma A. Electron shuttles in biotechnology. Curr Opin Biotechnol 2009;20:633e41.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Unveiling characteristics of dye-bearing microbial fuel cells for energy and materials recycling: Redox mediators Bor-Yann Chen a,*, Chung-Chuan Hsueh a, Shi-Qi Liu a, Jhao Yin Hung a, Yan Qiao b, Pei-Lin Yueh a, Yu-Min Wang a a b
Department of Chemical and Materials Engineering, National I-Lan University, I-Lan 26047, Taiwan Institute for Clean Energy & Advanced Materials, Southwest University, Chongqing 400715, China
article info
abstract
Article history:
This study disclosed why and how some decolorized intermediates (e.g., 2-aminophenol)
Received 26 December 2012
could act as electron-shuttling mediator(s) to enhance the capabilities of reductive decol-
Received in revised form
orization and bioelectricity generation. It also selected several model auxochrome-
17 March 2013
containing compounds structurally associated to 2AP to explore how chemical structure
Accepted 25 March 2013
influenced the feasibility of possible electron shuttles for power producing capabilities in
Available online 21 April 2013
microbial fuel cells (MFCs). The selection criteria of electron-shuttling mediators were suggested for optimal reductive decolorization and bioelectricity generation in MFCs for
Keywords:
practical application.
Auxochrome
Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Electron shuttle
reserved.
Microbial fuel cell Reductive decolorization
1.
Introduction
As an energy-famished country e Taiwan, renewable energy is always being considered as a promising alternative to help reduce the reliance on fossil fuels. In fact, due to Taiwan’s abundant bioresources significant attentions of biomassbased energy (BBE) have been paid nowadays as one of priority resources of renewable energy, in particular after the unfolding crisis with Japan’s devastating earthquake, tsunami and crippled unclear reactor meltdown in March, 2011. Among all BBE sources, microbial fuel cells (MFCs) can use naturallyoccurring microbes as biocatalysts to extract biofuel energy from oxidation of organic matter in wastewater for sustainable development. Recently, Lay et al. [1] used starchcontaining wastewater collected from Taiwan’s textile industry as a potential bioenergy source for biohydrogen
production. In addition, MFCs could simultaneously expedite bioelectricity generation as well as pollutant degradation during wastewater treatment [2,3]. In fact, electrochemical activities of microorganisms [4] significantly influenced the performance of bioelectricity production in MFC through at least three mechanisms: electron shuttling cell-secreting mediators (e.g., phenazine, quinones), membrane-bound redox proteins (e.g., mobile electron carriers such as cytochromes), and conductive pili (or nanowires) (e.g., wired communities of Geobacter sulfurreducens, Shewanella oneidensis) [5]. Regarding dye-bearing wastewater treatment, Cao et al. [6] and Li et al. [7] presented pioneer works on simultaneous dye decolorization (ca. <5% not decolorized) and power generation (ca. max. power density w500e600 mW m2) using mixed consortia-bearing MFC systems. Recently, Chen et al. [8] uncovered microbial characteristics of simultaneous
* Corresponding author. Fax: þ886 39357025. E-mail addresses: [email protected], [email protected] (B.-Y. Chen). 0360-3199/$ e see front matter Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijhydene.2013.03.132
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bioelectricity generation and dye decolorization (SBG&DD) using pure biodecolorizers (e.g., Klebsiella pneumoniae, Proteus hauseri), suggesting that reductive decolorization and bioelectricity generation of biodecolorizers was competitive to each other. Moreover, Chen et al. [9] quantitatively revealed that electrons extracted from this electron-transport chain of bacterial decolorizer via respiratory complexes were provided for either bioelectricity generation or color removal. They also successfully used 2-aminophenol (2AP) as a model exogenous mediator to enhance the performance of SBG&DD of P. hauseri. However, the mysteries why some amine phenolic compounds (e.g., 2AP) could be used to skyrocket the efficiency of SBG&DD were still remained uncertain to be explored. To decipher the mysteries of electron-shuttling characteristics of amine phenolic compounds augmentation, this study conducted cyclic voltammetric (CV) inspections upon compounds with similar functional groups. Comparison upon CV profiles of these possible mediators clearly suggested why and how they could or could not play as a role of electron shuttle. This comparative assessment can provide selection criteria of candidate electron transfer mediator for optimal power generation in MFC. These operation guidelines could provide professionals promising criteria to examine the feasibility of SBG&DD in MFCs before they launch practically.
2.
Methods
2.1.
Electrochemical measurements
Cyclic voltammetry of amine phenolic compounds was performed using an electrochemical workstation (Jiehan 5600, Taiwan) at 10 mV s1 scan rate. The working, counter, and reference electrodes were, respectively, a glassy carbon electrode (0.07 cm2), platinum electrode (6.08 cm2), and a Hg/Hg2Cl2 electrode filled with saturated KCl(aq). The glassy carbon electrode (GCE, ID ¼ 3 mm; model CHI104, CH Instruments Inc., USA) was successively polished with 0.05 mm alumina polish and then rinsed with 0.5 M H2SO4 and deionized water before use. The experiments were performed in phosphate buffer solutions (pH ¼ 7.0) at 0.1 M and the solutions were purged with nitrogen for 15 min prior to analysis. The scanning rate was 10 mV s1 over the range from 0.4 to 0.6 V [5]. The redox potentials recorded as Hg/Hg2Cl2 reference electrode were corrected by 0.241 V (i.e., E0 of Hg/Hg2Cl2) to the standard hydrogen electrode (SHE).
2.2.
MFC construction and microbial cultures
Membrane-free air cathode single-chamber MFCs (refer to Ref. [10] for schematic setup of MFC) were constructed in cylindrical tubes made by polymethyl methacrylate (PMMA) (cell sizing ID ¼ 54 mm, L ¼ 95 mm) with the operating volume of ca. 220 mL. Porous carbon cloth (CeTech) (without waterproofing or catalyst) with a projected area of ca. 22.9 cm2 (i.e., p 2.72) on one side was used as anode electrodes. The air cathode was almost identical to the anode in size and consisted of a polytetrafluoroethylene (PTFE) diffusion layer (CeTech) on the air-facing side.
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The procedures of bacterial cultures for MFCs were described elsewhere [11]. Culture medium in MFCs used in the study (unit: g L1) is 0.2 LB (tryptone 2, yeast extract 1, sodium chloride 10). A loopful of bacterial seed taken from an isolated colony on a LB-streak plate (i.e., LB medium supplemented with Bacto agar 20 g L1) was precultured in 50 mL LB broth laden with 200 mg L1 reactive blue 160 (RBu160; CAS 71827-76-9) using 250 mL Erlenmeyer flask for 12 h overnight at 30 C, 125 rpm. Cultured cells were then collected after centrifugation at 2400 g for 10 min. After cell-free supernatant was discarded, the biomass was rinsed and mixed with DI/DD (deionized/distilled) water. Rinse-and-mix procedures were repeated twice to harvest residue-free 2 rinsed biomass and then 2 rinsed biomass was concentrated with DI/DD water in 5 mL for inoculation to MFC.
2.3.
Experimental operations
Acclimation step: To uncover the effect of amine phenolic compounds (B12d, 12db) for stimulating bioelectricity generation and dye decolorization, approximately 5 mL concentrated biomass was well-mixed with 2.5 mL sterilized 0.2 LB medium in MFCs for acclimatization. During serial acclimation, every 48 h approximately 5 mL cell broth was meticulously replaced by impulse injection of 5 mL fresh sterile 4.4 LB medium to maintain initial concentration at 0.2 LB. Note that “impulse injection” indicated that a fresh nutrient medium was supplemented immediately in a very short period of time [12]. Then, the steady-state output power generation of MFCs was approximately achieved after approx. 30 days acclimation [9]. Experimental step: To explore stimulating characteristics of B12d for bioelectricity generation, the batch-fed mode of MFC operation with impulse injection of energy substrate was carried out at 25 C every 100 h. That is, 5.0 mL of 8.8 LB broth laden with appropriate concentrations of amine phenolic compound 12db or B12d was supplemented to MFCs to maintain culture medium in 0.2 LB for inspection. Approx. 1 h after impulse injection of energy substrate, the supplemented medium was considered to be well-distributed in MFC and then electrochemical analysis of MFCs was conducted.
2.4.
Sudan black B staining
Several drops (ca. 10 mL) of 12e16 h LB-culture cell broth (selected from cultured bacteria in different MFCs) were fixed on a glass slide by applying heat and then stained with a 3% Sudan black B (SB) (w/v in 70% ethanol, Sigma) solution for 10 min. The slide was then immersed in xylene until completely decolorized. The sample was counterstained with safranin (5%, w/v, in distilled water, Sigma Inc.) for 10 s, twice washed with distilled water, and dried afterward. Once the slide was completely dried, several drops of immersion oil were directly added on cell sample for microscopic examination under optical microscopy (Leica, Tokyo, Japan) [13]. Cellular compartments shown in black blue and pink color under optical microscopic observation indicated possible PHA-containing and PHA-absent granules in cell samples, respectively.
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3.
Results and discussion
3.1.
Assessment upon electron-shuttling mediators
Prior study [12] mentioned that bioelectricity generation and reductive decolorization were both competitive to each other. To uncover the mysteries behind bioelectricity generation and dye decolorization, the capability of amine phenolic compound (e.g., isomeric aminophenols; 2, 3, or 4-AP) [14] to enhance reductive decolorization was quantitatively disclosed. In fact, electron-shuttles (i.e., redox mediators) were reported to have capabilities to be reversibly oxidized and reduced as an electron carrier in multiple redox reactions. As a matter of fact, prior studies [15,16] also confirmed that 2-aminophenol (2AP) and benezene-1,2-diol (B12d) were electron-shuttling mediators to enhance bioelectricity generation and reductive decolorization based upon various MFC data. However, why and how some aminophenol isomers were capable to be electron-shuttling mediators was still remained open for discussion. To disclose such mysteries of electron-mediating properties, cyclic voltammetric inspections upon several compounds with similar functional groups (1,2-, 1,4-diaminobenezene (12db, 14db), 3-,4aminobenzoic acid (3ABA, 4ABA), aniline-2-sulfonic acid (A2SA), aniline-4-sulfonic acid (A4SA)) were carried out. As shown in Fig. 1, evidently only 2AP and 4AP could show capabilities of quasi-reversibility of redox processes for a separation of peak potentials of electron-shuttling mediators [15]. Regarding exogenous electron-shuttling mediator, prior study [8] suggested that phenyl methadiamine produced via reductive decolorization could mediate electron transfer in anodic biofilm of P. hauseri for power generation in MFC [10]. As indicated, appropriate amount of 2AP supplemented could significantly enhance the performance of bioelectricity generation in MFC. The output voltages were gradually increased with respect to an increased concentration of 2AP, suggesting that current production of P. hauseri in MFC could be significantly stimulated by the supplementation of 2AP as an exogenous electron-transfer mediator. However, not all isomeric aminophenols could effectively work as redox mediators or electron shuttles for extracellular electron transfer from P. hauseri cells to the anode (e.g., the supplementation of 3AP). As shown in Fig. 1, the absence of reduction and oxidation peak potentials for 3AP in its CV profile led to low feasibility for 3AP to be an electron-shuttling mediator. Similar example of 1,3dihydroxybenzene (or resorcinol) [17] also showed that meta-isomer was not appropriate to be an electron shuttle. Wang et al. [17] mentioned that “the electro-oxidation reaction of resorcinol (or 1,3-dihydroxybenzene) (a meta-isomer) shows an irreversible process, because no corresponding reductive peak was observed on the cathodic branch.” In addition, cyclic voltammogram for the redox processes of 1 mM benzene-1,3-diol could not show the presence of the oxidation or reduction potential (data not shown). Moreover, although 4AP could show the presence of reduction and oxidation peak potentials in CV profile (Fig. 1), zero output voltage was still observed in 4AP-supplemented MFC due to significant toxicity of 4AP on P. hauseri as shown elsewhere [8,10]. This point also confirmed that a reduced dye (i.e., aromatic amine) with relatively less biotoxicity might be feasible
as an exogenous mediator to stimulate electron transfer to anodic biofilm in MFC (e.g., 2AP). In fact, 2AP was also found to be a possible electron-shuttling mediator to Gram-negative aerobic rod Acinetobacter sp. 72 and facultatively anaerobic Gram-negative rod Enterobacter sp. m30 seeded mixed culture MFC. However, the mysteries why 2AP could play as an electron-shuttling mediator was still needed to be disclosed for optimization of power generation in MFC.
3.2.
Electron-mediating mechanism
In fact, the reversible redox properties of 2AP and 4AP were due to the reasons as follows: The electron shuttle can be reversibly interchanged between its reduced and oxidized form for mediating electron-transfer processes involved in energy production. For example, the inter-conversion of hydroquinone (p-dihydroxybenzene; a reduced form) and quinone (cyclohexa-2,5-diene-1,4-dione; an oxidized form) through the formation of active radicals (e.g., RD2 and resonance forms RD1A, RD1B in Fig. 2) can be reversibly reoxidized and re-reduced (Fig. 2 as illustrated in Ref. [18]). When hydroxyl groups are present on the benzene ring of the compounds (e.g., 2AP, 4AP, hydroquinone), these molecules can express significant electron-shuttling capabilities for electricity generation. As a matter of fact, this was the reasoning why chemicals 14db, A2SA, A4SA, 4ABA, and 3ABA (except of 12db which was lack of hydroxyl groups) could not own such shuttling functions as electron-transport mediators in MFC. However, 12db could play as an electron shuttle to enhance bioelectricity generation, since 12db could be oxidized via dimerization to be 1,2-diaminophenazine (Fig. 4(A)) [19]. As a matter of fact, 1,2-diaminophenazine could also play as redox mediator (Fig. 4(B); [16,17]). In addition, due to acidic characteristics of hydroxyl groups (eOH) of phenols, the acidic hydrogen can be dissociated to obtain phenolate anions, and then phenolate anions will be favorably oxidized to form active radicals (e.g., RD1A, RD1B, and RD2 in Fig. 2). Apparently, these steps of free-radical formation are crucial steps to generate redox mediators to mediate electron-transfer processes for energy and/or electricity production. For example, many derivatives of naphthoquinones or anthraquinones with the free-radical forming characteristics were also capable to be redox mediators [20,21]. In contrast, other functional groups (e.g., carboxyl (eCOOH) and sulfo (eSO3H) group) do not have such capabilities to play as a role of redox mediator. Although they owned an acidic hydrogen (Hþ) to be dissociated as an anion, they could not be easily oxidized to form free radicals afterward (e.g., RD1 in Fig. 2, RD3A, RD3B RD4A, and RD4B in Fig. 3(A, B)). Moreover, as hydrogen atom on the amino group (eNH2) of 14db is not acidic, it is thus not thermodynamically favorable to be deprotonated to form an anion. Thus, the compounds with amino group are not viable to be oxidized to form radical intermediates on the benzene ring (e.g., hydroquinone anion in Fig. 2) for electron transfer. That is, the compounds without hydroxy (eOH) group will not express electron-shuttling characteristics. Moreover, although all three AP isomers (i.e., o-, m-, p-isomers) contained hydroxyl groups, only 2 isomeric compounds
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Fig. 1 e CV profiles of model anxochrome-containing compounds in the study (N.D. denoted not determined).
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Fig. 2 e Proposed pathways of inter-conversion between hydroquinone and quinone.
(A)
(B)
(C)
Fig. 3 e (A) Proposed pathways of inter-conversion between 2AP and o-quinonimine, (B) Proposed pathways of interconversion between 4AP and p-quinonimine, (C) Proposed pathways of inter-conversion between 3AP and intermediates CI1-3.
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(A)
(B)
Fig. 4 e (A) Enzymatic dimerization of 1,2-diaminobenzene (12 db) to form electron shuttles. (B) Electron shuttling route for 1,2-diaminobenzene (12 db).
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2AP (o-isomer) and 4AP (p-isomer) could own electronshuttling capabilities. Similar to hydroquinone, 2AP and 4AP can be favorably oxidized to form o- and p-quinoniminium ion (i.e., iminium ion (¼NHþ2) containing chemical), and then be deprotonated to form imine (¼NH) containing o- and p-quinonimine structurally similar to electron shuttles 1,2benzoquinone (ortho-quinone) and 1,4-benzoquinone ( paraquinone), respectively, as shown in Fig. 3. However, due to characteristics of meta-isomer, 3AP cannot carry out such inter-conversion of reduced and oxidized form for electron-shuttling capabilities to mediate energy transfer in MFC. Since 3AP cannot carry a positive charge on benzene ring after oxidation, the positive charge will not be located on the carbon bonding to amino group as indicated in 2AP and 4AP (Fig. 3(A, B)). Thus, the positive charge at ortho or para position to the carbon that binds to amino group in oxidized 3AP (e.g., cationic intermediates CI1, CI2, CI3, CI4, and CI5 in Fig. 3(C)) will not be capable to form iminium ion. Similar to
Fig. 5 e (A) Optical microscopic observation upon possible PHA accumulation of bacterial cells of mixed consortia obtained from Aeromonas sp. C78, Aeromonas hydrophila NIU01, Acinetobacter johnsonii NIUx72, Proteus hauseri ZMd44, Enterobacter cancerogenus BYm30-seeded microbial fuel cells after Sudan-black staining (black colors (i.e., black-circled portions) and purple colors (i.e., red-circled portions) denoted (D) and (L) response of possible strains, respectively). (B) Comparison of dry cell weight, PHB concentration and PHB content of samples taken from Enterobacter cancerogenus BYm30, Proteus hauseri ZMd44, Acinetobacter johnsonii NIUx72, Aeromonas hydrophila NIU01 and Aeromonas sp. C78 seeded MFCs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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300
250
6th
15th 200
BYm30-M ZMd44-p NIUx72-M NIUx73-2 ZMd30-2 ZMd31-3 NIU01-M
200 150 100 50 0 0.0
[RBu160] (mg L-1)
[RBu160] (mg L-1)
250
BYm30-M ZMd44-p NIUx72-M NIUx73-2 ZMd30-2 ZMd31-3 NIU01-M
150
100
50
0 0.5
1.0
1.5
2.0
2.5
0.0
Time (hr)
0.5
1.0
1.5
2.0
2.5
Time (hr)
Fig. 6 e Time-series profiles of dye decolorization of different bacterial strain-seeded MFCs after 6th and 15th impulse injection of reactive blue 160 (RBu160). Strains BYm30, ZMd44, NIUx72, NIUx73, ZMd30, ZMd31 and NIU01 denoted Enterobacter cancerogenus, Proteus hauseri, Acinetobacter johnsonii, Aeromonas hydrophila, Klebsiella pneumoniae, Klebsiella pneumoniae and Aeromonas hydrophila, respectively.
aforementioned reasoning for 3-AP, 1,3-dihydroxybenzene could thus not be feasible as hydroquinone-like redox mediator [17]. Apparently, further deprotonation of oxidized 3AP to obtain imine as shown in 2AP and 4AP was not thermodynamically favorable. That is to say, 3AP can not play as a role of electron-shuttling mediator. These analyses uncovered the mysteries why and how only 2AP and 4AP can be electronshuttling mediators for energy recycling in MFC as mentioned herein.
3.3. MFCs
PHB accumulation and wastewater decolorization in
As aforementioned, mediator supplementation to microbial fuel cells could enhance energy-extraction through oxidation of organic matter in wastewater for energy recycling. As a matter of fact, MFC can also produce biopolymer during wastewater decolorization for simultaneous materials recycling. Here, optical microscopic observations upon bacterial cells of mixed cultures of Aeromonas sp. C78, Aeromonas hydrophila NIU01, Acinetobacter johnsonii NIUx72, P. hauseri ZMd44, Enterobacter cancerogenus BYm30 seeded mixed culture MFCs after SB staining indicated that significant black blue granules accumulated in these microbial cells in MFCs (Fig. 5). To uncover the profiles of PHA accumulation in MFCs, samples of these MFCs were taken for determining intracellular content of polyhydroxybutyrate (PHB) as an indicator
Table 1 e Tabulated list of initial decolorization rate (IDR) of various bacterium-seeded MFCs after 6th and 15th impulse injection of RBu160 (unit: mg LL1 hL1). MFC
IDR6
IDR15
BYm30-M ZMd44-p NIUx72-M NIUx73-2 ZMd30-2 ZMd31-3 NIU01-M
381.07 873.70 234.76 414.46 753.88 727.56 471.42
673.46 698.89 379.86 157.62 656.40 607.71 432.27
PHA. Fig. 6 indicated that only samples obtained from BYm30 and ZMd44-seeded MFCs contained significant amounts of PHB. This implied that those cultures were likely feasible candidate microorganisms for biodegradable polymer-polyhydroxyalkanoate (PHA) production. This finding showed that dye decolorization in parallel with bioelectricity generation and PHA production in MFC for energy and materials recycling was technically viable for sustainable development. Moreover, to reveal the feasibility of long-term stable simultaneous bioelectricity generation and reductive decolorization for MFC operation, dye decolorization profiles of several mixed culture-based MFCs were determined (Fig. 6, Table 1). As Table 1 indicated, after 6th and 15th impulse injection of reactive blue 160 (refer to Fig. 1 in Ref. [9] for operation procedure), color removal performance of MFCs still remained biologically stable. The points as aforementioned directly disclosed the promising feasibility of simultaneous energy and biomaterials recycling during wastewater decolorization.
4.
Conclusions
This study disclosed why and how some decolorized intermediates (e.g., 2-aminophenol) could act as electronshuttling mediator(s) to enhance the capabilities of reductive decolorization and bioelectricity generation. It also selected several model auxochrome-containing compounds structurally associated to 2AP to explore how chemical structure influenced the feasibility of possible electron shuttles for power producing capabilities in microbial fuel cells (MFCs). The selection criteria of electron-shuttling mediators were suggested for optimal reductive decolorization and bioelectricity generation in MFCs for practical application.
Acknowledgment The authors sincerely appreciate financial support (NSC 982221-E-197-007-MY3 and NSC 100-2621-M-197-001) from
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Taiwan’s National Science Council for the project of Microbial Fuel Cell (MFC)sdg conducted in Biochemical Engineering Lab, NIU.
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