Bioresource Technology 192 (2015) 821–825
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Microbial surface displayed enzymes based biofuel cell utilizing degradation products of lignocellulosic biomass for direct electrical energy Shuqin Fan a,b,1, Chuantao Hou a,1, Bo Liang a,b, Ruirui Feng a, Aihua Liu a,b,⇑ a Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China b University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China
h i g h l i g h t s BFC based electrical energy conversion from degradation products of biomass. Microbial surface displayed XDH and GDH used as anode catalysts. The BFC exhibiting high OCP of 0.80 V and maximal power density of 53 lW cm
2
.
Acceptable operational stability and energy conversion efficiency.
a r t i c l e
i n f o
Article history: Received 24 April 2015 Received in revised form 23 May 2015 Accepted 25 May 2015 Available online 28 May 2015 Keywords: Enzymatic biofuel cell Degradation products of lignocellulosic biomass Xylose dehydrogenase displayed bacteria Glucose dehydrogenase displayed bacteria Direct electrical energy conversion
a b s t r a c t In this work, a bacterial surface displaying enzyme based two-compartment biofuel cell for the direct electrical energy conversion from degradation products of lignocellulosic biomass is reported. Considering that the main degradation products of the lignocellulose are glucose and xylose, xylose dehydrogenase (XDH) displayed bacteria (XDH-bacteria) and glucose dehydrogenase (GDH) displayed bacteria (GDH-bacteria) were used as anode catalysts in anode chamber with methylene blue as electron transfer mediator. While the cathode chamber was constructed with laccase/multi-walled-carbon nanotube/glas sy-carbon-electrode. XDH-bacteria exhibited 1.75 times higher catalytic efficiency than GDH-bacteria. This assembled enzymatic fuel cell exhibited a high open-circuit potential of 0.80 V, acceptable stability and energy conversion efficiency. Moreover, the maximum power density of the cell could reach 53 lW cm2 when fueled with degradation products of corn stalk. Thus, this finding holds great potential to directly convert degradation products of biomass into electrical energy. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Biomass has been shown as a feasible alternative green energy to fossil fuels. Lignocellulose exists extensively within agricultural and agro-industrial residues such as nut shells, corn cobs, grasses, wheat straw, leaves, swine solid cattle manure, and so on. Lignocellulosic biomass represents one of the most promising renewable resources that can be utilized for the biological production of biofuel (Liu et al., 2014; Sun and Cheng, 2002). The contents of cellulose,
⇑ Corresponding author at: Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China. Tel.: +86 532 80662758; fax: +86 532 80662778. E-mail address: [email protected] (A. Liu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biortech.2015.05.090 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
hemicellulose, and lignin change over different agricultural residues (Sun and Cheng, 2002). The ratios of reducing sugars are varying with the sources of lignocelluloses and pretreatment process of lignocellulosic biomass, however, the main reducing sugars in hydrolysates are glucose and xylose (Zhang et al., 2014). To date, lignocellulose hydrolyzates have been widely used for the co-fermentation of xylose and glucose to ethanol (Cheng et al., 2015; Madhavan et al., 2012), which could effectively improve the utilization rate of raw materials, and increase the productivity of ethanol and reduce their costs. These meaningful researches greatly improved energy conversion efficiency, reduced costs and promised for the great potential to practical application of degradation products of lignocellulose. However, the yield of ethanol is not very ideal, so it is imperative to find another way for the utilization of lignocellulose degradation products.
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Biofuel cells (BFCs) utilize electricigens or enzymes as biocatalysts for the conversion of electrical energy directly from a chemical reaction (Yan et al., 2006). Enzymes as catalysts in fuel cells performed better than conventional catalysts in terms of the higher specificity and rates of reaction (Leech et al., 2012). Most enzymatic biofuel cells are capable of operating at neutral pH and physiological temperature, easily miniaturizing due to membraneless, which are well suitable for implanted or portable devices (Heller and Feldman, 2008; Moehlenbrock and Minteer, 2008; Szczupak et al., 2012a). These BFCs have environmental advantages over combustion for without NOx production and higher efficiency (Cracknell et al., 2008). However, for some enzymes such as glucose oxidase and laccase, their activity sites are buried within the protein, making the electron transfer to electrode surface difficult (Leech et al., 2012; Moehlenbrock and Minteer, 2008; Osman et al., 2011). What is more, although BFCs have a long history in the literature, long-time stability is still serious problem that hampers biofuel cell successful development. The catalytic activity of enzyme is determined by the three-dimensional protein structure, which is affected by temperature, pH and chemical components of the solution environment (Barton et al., 2004; Moehlenbrock and Minteer, 2008). Moreover, the cost of enzyme purification could block the practical application of enzyme biofuel cells. In comparison with pure enzymes, bacteria surface displaying of redox enzymes exhibited improved stability and cell performance without complex enzyme purification process. The previous studies showed that, xylose dehydrogenase (XDH) displayed bacteria (XDH-bacteria) and glucose dehydrogenase (GDH) displayed bacteria (GDH-bacteria) exhibited higher stability than the pure free counterparts (Liang et al., 2013, 2012). In this paper, for the first time, novel two-compartment BFCs were constructed using both XDH-bacteria and GDH-bacteria as anode catalysts as well as degradation products of biomass as fuel. The cathode chamber was provided with laccase to catalyze the reduction of oxygen. This assembled enzymatic fuel cell exhibited a high open-circuit potential of 0.80 V, acceptable stability and energy conversion efficiency. A maximum output power density of about 53 lW cm2 was obtained when the degradation liquid of corn stalk was used as fuel. 2. Methods 2.1. Preparation of modified bioanode and biocathode The glassy carbon electrode (GCE, diameter of 3 mm) was polished to a mirror finish using 0.3 and 0.05 lm alumina slurry, followed by sonicating in anhydrous ethanol and water, respectively. Finally, the electrode was thoroughly rinsed with deionized water and dried at room temperature. A 4 lL of multiwalled carbon nanotubes (MWNTs) dispersion was cast on the inverted electrode, and dried in air. Then 5 lL of Nafion solution (0.05 wt%) was syringed to the electrode surface. The as-prepared electrode is denoted as Nafion/MWNTs/GCE. For the preparation of biocathode, 4 lL laccase (10 mg mL1) aqueous dispersion was coated on the MWNTs/GCE and then cross-linked with 2 lL of glutaraldehyde (1 wt%), finally dried in the fridge (4 °C) overnight. The thus-prepared electrode is denoted as laccase/MWNTs/GCE.
4.5) containing 0.5 mM of 2,20 -azinobis (3-ethylbenzothiazoli ne-6-sulfonic acid) diammonium salt (ABTS). The anolytes were 0.1 M PBS buffer (pH 7.4) containing 10 mM NAD+ (the oxidized form of nicotinamide adenine dinucleotide), 0.5 mM methylene blue (MB) and XDH-bacteria with varying xylose conc., 0.1 M PBS buffer (pH 7.4) containing 10 mM NAD+, 0.5 mM MB and GDH-bacteria with varying glucose conc., and 0.1 M PBS buffer (pH 7.4) containing 10 mM NAD+, 0.5 mM MB and XDH-bacteria and GDH-bacteria with lignocellulose degradation products for the xylose/O2, glucose/O2 and lignocellulose degradation products/O2 BFC, separately. 3. Results and discussions 3.1. Electrocatalytic oxidation of xylose with XDH-bacteria and glucose with GDH-bacteria Previously, highly sensitive D-xylose and D-glucose electrochemical biosensor based on XDH-bacteria and GDH-bacteria, respectively were constructed (Li et al., 2012; Liang et al., 2013). Xylose (or glucose) could be catalytically oxidized by XDH (or GDH) with NAD+ as the coenzyme, which is reduced to NADH. Then the generated NADH would be oxidized further into NAD+ on the electrode surface. Considering that the products are mainly glucose and xylose after the hydrolysis of lignocellulose (Zhang et al., 2014), the electrocatalytic oxidation of xylose and glucose with the XDH-bacteria and GDH-bacteria in solution were investigated, respectively. MWNTs was used to facilitate electron transfer rate and enhance the catalytic activity. As an energy-production, an enzymatic BFC generates maximum power, meaning both high potential and high current (Barton et al., 2004). Generally, for a given biofuel cell, the open-circuit voltage is determined by the onset potential for catalysis at the cathode and anode (Barton et al., 2004; Hou et al., 2014). Therefore, MB was used as electron transfer mediator and for decreasing the onset potential of the xylose or glucose electrocatalysis. Initially, to evaluate the effect of the fuel type and mediator of MB on the performance of electrode, the electrochemical behaviors of Nafion/MWNTs/GCE in two separate experiments using either xylose/XDH-bacteria or glucose/GDH-bacteria were examined. A couple of sensitive and reversible redox peaks around 0.3 V (vs. SCE) were obtained from the cyclic voltammograms (CVs) (Fig. 1A and B, curve a). The bioelectrocatalytic oxidation of xylose and glucose in the presence of 0.5 mM MB in solution was occurred at a low potential of about 0.25 V. It should be noted here, the anodic currents for both cases were almost the same, however, the xylose conc. (3 mM) was 1.33 times lower than that of glucose (7 mM), suggesting that the NADH produced by XDH-bacteria was more than that of GDH-bacteria. This result was consistent well with that the Vmax value of XDH-bacteria was 2.75 times of that value of GDH-bacteria (Fig. S1, Supplementary materials). The polarization curves were recorded in different concentrations of xylose and glucose in the presence of 0.5 mM MB and 5 mM NAD+, respectively (Fig. 1C and D). The oxidation of xylose and glucose were observed at 0.25 V, which were in agreement with CVs. The electrocatalytic current improved significantly with the increase in concentration from 1 to 10 mM for xylose and 4–20 mM for glucose, and reached 0.138 mA cm2 and 0.075 mA cm2 at 0.2 V in 10 mM xylose and 20 mM glucose solution, respectively.
2.2. Assembly of biofuel cell 3.2. Characterization of the biocathode The xylose/O2, glucose/O2 and lignocellulose degradation products/O2 biofuel cells were assembled in two-compartment separately. The cation exchange membrane was used to separate the anodic and cathodic compartments. The catholyte was oxygen-saturated 0.2 M citric acid–Na2HPO4 buffer solution (pH
Laccase is a specific enzyme for catalyzing a 4-electron reduction of O2 at high potential, which has been extensively used as the cathode biocatalyst in enzymatic BFCs. However, laccase species are essentially inactive at pH 7 and have optimal activity in
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Fig. 1. (A) CV curves of the MWNTs/GCE in (a) 0.2 M phosphate solution (pH 7.4) containing 5 mM NAD+, 0.5 mM MB, XDH-displayed E. coli and in (b) 0.2 M phosphate solution containing 3 mM xylose, 5 mM NAD+, 0.5 mM MB and XDH-displayed E. coli. Scan rate, 50 mV s1. (B) CVs of the Nafion/MWNTs/GCE in (a) 0.2 M phosphate solution (pH 7.4) containing 5 mM NAD+, 0.5 mM MB, GDH-displayed E. coli and in (b) 0.2 M phosphate solution containing 7 mM glucose, 5 mM NAD+, 0.5 mM MB and GDHdisplayed E. coli. Scan rate, 50 mV s1. (C) Polarization curves of the anode in presence of XDH-displayed E. coli. Xylose concentration: 1 (a), 4 (b) and 10 mM (c) in phosphate solution containing 5 mM NAD+. Scan rate, 1 mVs1 (D) Polarization curves of the anode in presence of GDH-displayed E. coli. Glucose concentration: 4 (a), 10 (b) and 20 mM (c) in phosphate solution (pH 7.4) containing 5 mM NAD+. Scan rate, 1 mV s1.
the pH 4–5 range (Barton et al., 2004). Thus, the electrocatalytic performance of the modified laccase/MWNTs/GCE biocathode was evaluated by electrochemical measurements in citric acid– Na2HPO4 buffer (pH 4.5) solution containing 0.5 mM ABTS. A couple of quasi-reversible redox process with anodic peak at 0.38 V and cathodic peak at 0.53 V was observed in the presence of 0.5 mM ABTS under N2 saturated condition (Fig. 2A, curve b). In the presence of O2, an increased reduction current appeared while the oxidation peak of the ABTS almost disappeared (Fig. 2A, curve c), suggesting that the catalytic reduction of O2 by laccase with ABTS as the electron mediator was realized. The polarization curves of the laccase/MWNTs/GCE biocathode toward O2 reduction showed that the electrocatalytic reduction of O2 started at about 0.58 V (Fig. 2B), which were compliant with CVs.
the cathode (0.6 V) (Szczupak et al., 2012b). For a given BFC, the measured OCP is determined by the difference between the onset potential for catalysis at the anode and cathode (Cracknell et al., 2008). In this paper, all BFCs were assembled with the same biocathode and the oxidation of xylose and glucose showed the same onset potential (Fig. 1). Higher OCP indicated the electrodes operated closer to the thermodynamic potential with lower overpotential (Cracknell et al., 2008). The power density of the BFC was influenced by the fuel concentrations. The cell output power density was nearly linear with the increasing xylose or glucose concentration, and thereafter a plateau reached when the fuel concentration was higher than 10 mM xylose for xylose/O2 BFC and 20 mM glucose for glucose/O2 BFC, respectively (Fig. 3B).
3.4. The lignocellulose degradation products/oxygen BFC 3.3. Characterization of the xylose/oxygen and glucose/oxygen BFCs Before construction of the degradation products of lignocellulose/oxygen BFC, the xylose/oxygen and glucose/oxygen BFCs were studied separately. The maximal power output density for the xylose/oxygen BFC was 42.0 lW cm2 at 0.41 V (Fig. 3A, curve a). Similarly, the maximal power output density for the glucose/oxygen BFC was 16.3 lW cm2 at 0.44 V (Fig. 3A, curve b). That is, the power density of xylose/oxygen BFC was 1.5 times higher than that of glucose/oxygen BFC under the same fuel concentration. Both BFCs showed the same high OCP of 0.80 V (Fig. 3A), which was higher than those values reported for glucose/air BFC (0.75 V) (Wen et al., 2011) and BFC with BOD-displaying yeast in
The proposed BFC was applied by using two kinds of degradation products of lignocelluloses as fuels. The samples were diluted with PBS buffer solution (pH 7.4) to fit into the appropriate concentration range according to the above experimental results. The xylose and glucose concentrations in the degradation products were measured by liquid chromatography method. As controls, the standard xylose and glucose mixed solution with the same concentration of those in the degradation products were prepared, and the power densities were measured, respectively. For enzymatic hydrolysis coupled with physico-chemical process based biomass fuels, the power densities of the cell were very similar to those values when using standard mixed glucose/xylose solutions (Table 1).
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Fig. 2. (A) CVs of the laccase/MWNTs/GCE biocathode in 0.2 M phosphate–citric acid buffer (pH 4.5) (a) without ABTS under N2-saturated atmosphere, (b) in the presence of 0.5 mM ABTS under N2-saturated and (c) in the presence of 0.5 mM ABTS under oxygen-saturated atmosphere. Scan rate, 20 mV s1. (B) Polarization curves of the laccase/MWNTs/GCE biocathode in 0.2 M phosphate–citric acid buffer (pH 4.5) (a) in the presence of 0.5 mM ABTS under N2-saturated atmosphere, (b) in the presence of 0.5 mM ABTS under ambient air, and (c) in the presence of 0.5 mM ABTS under oxygen-saturated atmosphere. Scan rate, 1 mV s1.
3.5. Stability of the BFC As the energy device, the lifetime is of great importance. Enzymatic biofuel cells suffer from an inevitable disadvantage for long-term operation, because of the loss of enzyme activity. To test the long-term stability of the assembled BFC, the cell was recorded continuously in as-prepared degradation products solution containing 10 mM NAD+ under ambient air. The power density increased during the first 3 h recording, after that, it began to decrease gradually (Fig. S2, Supplementary materials). Similar phenomena were observed in previous report (Yan et al., 2006). The exact reason for this is unclear for the moment. It is probably originating from the accumulation of the concentration of NADH during the beginning process. After 10 h operation, it still remained 80% its original maximal power (Fig. S2), indicating a favorably stable power output process, which may contribute to the improved stability of the mutant XDH and mutant GDH displayed on bacterial surface (Liang et al., 2013). 3.6. Faraday efficiency Faraday efficiency is an important parameter of a BFC, which is defined as the ratio of coulombs (amperes seconds) transferred from the substrate to the anode, to the theoretical maximum coulombs produced provided that all of the substrate in the cell
Fig. 3. (A) Dependence of the power density on the cell voltage: (a), xylose/O2 BFC, the anolyte was 0.1 M PBS buffer (pH 7.4) containing 15 mM xylose, 10 mM NAD+, 0.5 mM MB and XDH-bacteria; (b), glucose/O2 BFC, the anolyte was 0.1 M PBS buffer (pH 7.4) containing 25 mM glucose, 10 mM NAD+, 0.5 mM MB and GDHbacteria. (B) Dependence of the power density on the sugar fuel concentration: (a), xylose/O2 BFC; (b), glucose/O2 BFC.
Table 1 The power density using different degradation products of lignocellulose as fuels. Lignocellulose
Sugar conc. in the degradation products
Power density (lW cm2)c,d
Power density (lW cm2)d,e
Corn cob 1#a
Xylose (8.3 mM), glucose (19.7 mM) Xylose (8.6 mM), glucose (21.3 mM)
45.1 ± 3.8
48.5 ± 3.3
53.2 ± 1.9
52.4 ± 4.1
Corn stalk 2#b
a Corn cob 1#, the degradation products of corn cob, first treated with enzymatic hydrolysis and followed by physico-chemical process. b Corn stalk 2#, the degradation products of corn stalk, first treated with enzymatic hydrolysis, and followed by physico-chemical process. c The two-compartment degradation products of lignocellulose/O2 BFCs were assembled. The anolyte: 0.1 M PBS buffer (pH 7.4) containing 10 mM NAD+, 0.5 mM MB and XDH-bacteria and GDH-bacteria and lignocellulose degradation products. d The power density is expressed as the mean ± standard deviation for three separate experiments. e The two-compartment was the same as that of degradation products of lignocellulose/O2 BFCs. The anolyte: 0.1 M PBS buffer (pH 7.4) containing 10 mM NAD+, 0.5 mM MB and XDH-bacteria and GDH-bacteria with the mixture of standard xylose and glucose as fuels in the same concentration as in the degradation products.
is oxidized (Osman et al., 2011). This power production was accompanied by the consumption of diluted lignocellulose degradation products. The Faraday efficiency in this work was about 41% (Fig. 4), which was higher than that value of 30% for
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References
Fig. 4. Profiles for current generation (a) and cumulative Faraday efficiency of the assembled BFC (b).
glucose/O2 biofuel cell (Velasquez-Orta et al., 2011). Generally, two-compartment may lead to higher power outputs with low faraday efficiencies (Osman et al., 2011). 4. Conclusions The degradation products of lignocellulosic biomass/O2 BFC in two-compartment was fabricated for the first time, which exhibited a maximum power density of 53 lW cm2 with degradation products of corn stalk as fuel. Moreover, the assembled BFC exhibited a high open-circuit potential of 0.80 V, acceptable stability and energy conversion efficiency. It is envisioned that the assembled BFC is promising to use lignocellulosic biomass as source energy. Acknowledgements This work was financially supported by National Natural Science Foundation of China (Nos. 91227116, 21275152, and 21475144), the Postdoctoral Science Foundation of China (No. 2014M560586), Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences (CASKLB201505) and the Qingdao Institute of Bioenergy and Bioprocess Technology Director Innovation Foundation for Young Scientists (Y37203210S). 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.2015.05. 090.
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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Microbial surface displayed enzymes based biofuel cell utilizing degradation products of lignocellulosic biomass for direct electrical energy Shuqin Fan a,b,1, Chuantao Hou a,1, Bo Liang a,b, Ruirui Feng a, Aihua Liu a,b,⇑ a Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China b University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China
h i g h l i g h t s BFC based electrical energy conversion from degradation products of biomass. Microbial surface displayed XDH and GDH used as anode catalysts. The BFC exhibiting high OCP of 0.80 V and maximal power density of 53 lW cm
2
.
Acceptable operational stability and energy conversion efficiency.
a r t i c l e
i n f o
Article history: Received 24 April 2015 Received in revised form 23 May 2015 Accepted 25 May 2015 Available online 28 May 2015 Keywords: Enzymatic biofuel cell Degradation products of lignocellulosic biomass Xylose dehydrogenase displayed bacteria Glucose dehydrogenase displayed bacteria Direct electrical energy conversion
a b s t r a c t In this work, a bacterial surface displaying enzyme based two-compartment biofuel cell for the direct electrical energy conversion from degradation products of lignocellulosic biomass is reported. Considering that the main degradation products of the lignocellulose are glucose and xylose, xylose dehydrogenase (XDH) displayed bacteria (XDH-bacteria) and glucose dehydrogenase (GDH) displayed bacteria (GDH-bacteria) were used as anode catalysts in anode chamber with methylene blue as electron transfer mediator. While the cathode chamber was constructed with laccase/multi-walled-carbon nanotube/glas sy-carbon-electrode. XDH-bacteria exhibited 1.75 times higher catalytic efficiency than GDH-bacteria. This assembled enzymatic fuel cell exhibited a high open-circuit potential of 0.80 V, acceptable stability and energy conversion efficiency. Moreover, the maximum power density of the cell could reach 53 lW cm2 when fueled with degradation products of corn stalk. Thus, this finding holds great potential to directly convert degradation products of biomass into electrical energy. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Biomass has been shown as a feasible alternative green energy to fossil fuels. Lignocellulose exists extensively within agricultural and agro-industrial residues such as nut shells, corn cobs, grasses, wheat straw, leaves, swine solid cattle manure, and so on. Lignocellulosic biomass represents one of the most promising renewable resources that can be utilized for the biological production of biofuel (Liu et al., 2014; Sun and Cheng, 2002). The contents of cellulose,
⇑ Corresponding author at: Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China. Tel.: +86 532 80662758; fax: +86 532 80662778. E-mail address: [email protected] (A. Liu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biortech.2015.05.090 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
hemicellulose, and lignin change over different agricultural residues (Sun and Cheng, 2002). The ratios of reducing sugars are varying with the sources of lignocelluloses and pretreatment process of lignocellulosic biomass, however, the main reducing sugars in hydrolysates are glucose and xylose (Zhang et al., 2014). To date, lignocellulose hydrolyzates have been widely used for the co-fermentation of xylose and glucose to ethanol (Cheng et al., 2015; Madhavan et al., 2012), which could effectively improve the utilization rate of raw materials, and increase the productivity of ethanol and reduce their costs. These meaningful researches greatly improved energy conversion efficiency, reduced costs and promised for the great potential to practical application of degradation products of lignocellulose. However, the yield of ethanol is not very ideal, so it is imperative to find another way for the utilization of lignocellulose degradation products.
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Biofuel cells (BFCs) utilize electricigens or enzymes as biocatalysts for the conversion of electrical energy directly from a chemical reaction (Yan et al., 2006). Enzymes as catalysts in fuel cells performed better than conventional catalysts in terms of the higher specificity and rates of reaction (Leech et al., 2012). Most enzymatic biofuel cells are capable of operating at neutral pH and physiological temperature, easily miniaturizing due to membraneless, which are well suitable for implanted or portable devices (Heller and Feldman, 2008; Moehlenbrock and Minteer, 2008; Szczupak et al., 2012a). These BFCs have environmental advantages over combustion for without NOx production and higher efficiency (Cracknell et al., 2008). However, for some enzymes such as glucose oxidase and laccase, their activity sites are buried within the protein, making the electron transfer to electrode surface difficult (Leech et al., 2012; Moehlenbrock and Minteer, 2008; Osman et al., 2011). What is more, although BFCs have a long history in the literature, long-time stability is still serious problem that hampers biofuel cell successful development. The catalytic activity of enzyme is determined by the three-dimensional protein structure, which is affected by temperature, pH and chemical components of the solution environment (Barton et al., 2004; Moehlenbrock and Minteer, 2008). Moreover, the cost of enzyme purification could block the practical application of enzyme biofuel cells. In comparison with pure enzymes, bacteria surface displaying of redox enzymes exhibited improved stability and cell performance without complex enzyme purification process. The previous studies showed that, xylose dehydrogenase (XDH) displayed bacteria (XDH-bacteria) and glucose dehydrogenase (GDH) displayed bacteria (GDH-bacteria) exhibited higher stability than the pure free counterparts (Liang et al., 2013, 2012). In this paper, for the first time, novel two-compartment BFCs were constructed using both XDH-bacteria and GDH-bacteria as anode catalysts as well as degradation products of biomass as fuel. The cathode chamber was provided with laccase to catalyze the reduction of oxygen. This assembled enzymatic fuel cell exhibited a high open-circuit potential of 0.80 V, acceptable stability and energy conversion efficiency. A maximum output power density of about 53 lW cm2 was obtained when the degradation liquid of corn stalk was used as fuel. 2. Methods 2.1. Preparation of modified bioanode and biocathode The glassy carbon electrode (GCE, diameter of 3 mm) was polished to a mirror finish using 0.3 and 0.05 lm alumina slurry, followed by sonicating in anhydrous ethanol and water, respectively. Finally, the electrode was thoroughly rinsed with deionized water and dried at room temperature. A 4 lL of multiwalled carbon nanotubes (MWNTs) dispersion was cast on the inverted electrode, and dried in air. Then 5 lL of Nafion solution (0.05 wt%) was syringed to the electrode surface. The as-prepared electrode is denoted as Nafion/MWNTs/GCE. For the preparation of biocathode, 4 lL laccase (10 mg mL1) aqueous dispersion was coated on the MWNTs/GCE and then cross-linked with 2 lL of glutaraldehyde (1 wt%), finally dried in the fridge (4 °C) overnight. The thus-prepared electrode is denoted as laccase/MWNTs/GCE.
4.5) containing 0.5 mM of 2,20 -azinobis (3-ethylbenzothiazoli ne-6-sulfonic acid) diammonium salt (ABTS). The anolytes were 0.1 M PBS buffer (pH 7.4) containing 10 mM NAD+ (the oxidized form of nicotinamide adenine dinucleotide), 0.5 mM methylene blue (MB) and XDH-bacteria with varying xylose conc., 0.1 M PBS buffer (pH 7.4) containing 10 mM NAD+, 0.5 mM MB and GDH-bacteria with varying glucose conc., and 0.1 M PBS buffer (pH 7.4) containing 10 mM NAD+, 0.5 mM MB and XDH-bacteria and GDH-bacteria with lignocellulose degradation products for the xylose/O2, glucose/O2 and lignocellulose degradation products/O2 BFC, separately. 3. Results and discussions 3.1. Electrocatalytic oxidation of xylose with XDH-bacteria and glucose with GDH-bacteria Previously, highly sensitive D-xylose and D-glucose electrochemical biosensor based on XDH-bacteria and GDH-bacteria, respectively were constructed (Li et al., 2012; Liang et al., 2013). Xylose (or glucose) could be catalytically oxidized by XDH (or GDH) with NAD+ as the coenzyme, which is reduced to NADH. Then the generated NADH would be oxidized further into NAD+ on the electrode surface. Considering that the products are mainly glucose and xylose after the hydrolysis of lignocellulose (Zhang et al., 2014), the electrocatalytic oxidation of xylose and glucose with the XDH-bacteria and GDH-bacteria in solution were investigated, respectively. MWNTs was used to facilitate electron transfer rate and enhance the catalytic activity. As an energy-production, an enzymatic BFC generates maximum power, meaning both high potential and high current (Barton et al., 2004). Generally, for a given biofuel cell, the open-circuit voltage is determined by the onset potential for catalysis at the cathode and anode (Barton et al., 2004; Hou et al., 2014). Therefore, MB was used as electron transfer mediator and for decreasing the onset potential of the xylose or glucose electrocatalysis. Initially, to evaluate the effect of the fuel type and mediator of MB on the performance of electrode, the electrochemical behaviors of Nafion/MWNTs/GCE in two separate experiments using either xylose/XDH-bacteria or glucose/GDH-bacteria were examined. A couple of sensitive and reversible redox peaks around 0.3 V (vs. SCE) were obtained from the cyclic voltammograms (CVs) (Fig. 1A and B, curve a). The bioelectrocatalytic oxidation of xylose and glucose in the presence of 0.5 mM MB in solution was occurred at a low potential of about 0.25 V. It should be noted here, the anodic currents for both cases were almost the same, however, the xylose conc. (3 mM) was 1.33 times lower than that of glucose (7 mM), suggesting that the NADH produced by XDH-bacteria was more than that of GDH-bacteria. This result was consistent well with that the Vmax value of XDH-bacteria was 2.75 times of that value of GDH-bacteria (Fig. S1, Supplementary materials). The polarization curves were recorded in different concentrations of xylose and glucose in the presence of 0.5 mM MB and 5 mM NAD+, respectively (Fig. 1C and D). The oxidation of xylose and glucose were observed at 0.25 V, which were in agreement with CVs. The electrocatalytic current improved significantly with the increase in concentration from 1 to 10 mM for xylose and 4–20 mM for glucose, and reached 0.138 mA cm2 and 0.075 mA cm2 at 0.2 V in 10 mM xylose and 20 mM glucose solution, respectively.
2.2. Assembly of biofuel cell 3.2. Characterization of the biocathode The xylose/O2, glucose/O2 and lignocellulose degradation products/O2 biofuel cells were assembled in two-compartment separately. The cation exchange membrane was used to separate the anodic and cathodic compartments. The catholyte was oxygen-saturated 0.2 M citric acid–Na2HPO4 buffer solution (pH
Laccase is a specific enzyme for catalyzing a 4-electron reduction of O2 at high potential, which has been extensively used as the cathode biocatalyst in enzymatic BFCs. However, laccase species are essentially inactive at pH 7 and have optimal activity in
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Fig. 1. (A) CV curves of the MWNTs/GCE in (a) 0.2 M phosphate solution (pH 7.4) containing 5 mM NAD+, 0.5 mM MB, XDH-displayed E. coli and in (b) 0.2 M phosphate solution containing 3 mM xylose, 5 mM NAD+, 0.5 mM MB and XDH-displayed E. coli. Scan rate, 50 mV s1. (B) CVs of the Nafion/MWNTs/GCE in (a) 0.2 M phosphate solution (pH 7.4) containing 5 mM NAD+, 0.5 mM MB, GDH-displayed E. coli and in (b) 0.2 M phosphate solution containing 7 mM glucose, 5 mM NAD+, 0.5 mM MB and GDHdisplayed E. coli. Scan rate, 50 mV s1. (C) Polarization curves of the anode in presence of XDH-displayed E. coli. Xylose concentration: 1 (a), 4 (b) and 10 mM (c) in phosphate solution containing 5 mM NAD+. Scan rate, 1 mVs1 (D) Polarization curves of the anode in presence of GDH-displayed E. coli. Glucose concentration: 4 (a), 10 (b) and 20 mM (c) in phosphate solution (pH 7.4) containing 5 mM NAD+. Scan rate, 1 mV s1.
the pH 4–5 range (Barton et al., 2004). Thus, the electrocatalytic performance of the modified laccase/MWNTs/GCE biocathode was evaluated by electrochemical measurements in citric acid– Na2HPO4 buffer (pH 4.5) solution containing 0.5 mM ABTS. A couple of quasi-reversible redox process with anodic peak at 0.38 V and cathodic peak at 0.53 V was observed in the presence of 0.5 mM ABTS under N2 saturated condition (Fig. 2A, curve b). In the presence of O2, an increased reduction current appeared while the oxidation peak of the ABTS almost disappeared (Fig. 2A, curve c), suggesting that the catalytic reduction of O2 by laccase with ABTS as the electron mediator was realized. The polarization curves of the laccase/MWNTs/GCE biocathode toward O2 reduction showed that the electrocatalytic reduction of O2 started at about 0.58 V (Fig. 2B), which were compliant with CVs.
the cathode (0.6 V) (Szczupak et al., 2012b). For a given BFC, the measured OCP is determined by the difference between the onset potential for catalysis at the anode and cathode (Cracknell et al., 2008). In this paper, all BFCs were assembled with the same biocathode and the oxidation of xylose and glucose showed the same onset potential (Fig. 1). Higher OCP indicated the electrodes operated closer to the thermodynamic potential with lower overpotential (Cracknell et al., 2008). The power density of the BFC was influenced by the fuel concentrations. The cell output power density was nearly linear with the increasing xylose or glucose concentration, and thereafter a plateau reached when the fuel concentration was higher than 10 mM xylose for xylose/O2 BFC and 20 mM glucose for glucose/O2 BFC, respectively (Fig. 3B).
3.4. The lignocellulose degradation products/oxygen BFC 3.3. Characterization of the xylose/oxygen and glucose/oxygen BFCs Before construction of the degradation products of lignocellulose/oxygen BFC, the xylose/oxygen and glucose/oxygen BFCs were studied separately. The maximal power output density for the xylose/oxygen BFC was 42.0 lW cm2 at 0.41 V (Fig. 3A, curve a). Similarly, the maximal power output density for the glucose/oxygen BFC was 16.3 lW cm2 at 0.44 V (Fig. 3A, curve b). That is, the power density of xylose/oxygen BFC was 1.5 times higher than that of glucose/oxygen BFC under the same fuel concentration. Both BFCs showed the same high OCP of 0.80 V (Fig. 3A), which was higher than those values reported for glucose/air BFC (0.75 V) (Wen et al., 2011) and BFC with BOD-displaying yeast in
The proposed BFC was applied by using two kinds of degradation products of lignocelluloses as fuels. The samples were diluted with PBS buffer solution (pH 7.4) to fit into the appropriate concentration range according to the above experimental results. The xylose and glucose concentrations in the degradation products were measured by liquid chromatography method. As controls, the standard xylose and glucose mixed solution with the same concentration of those in the degradation products were prepared, and the power densities were measured, respectively. For enzymatic hydrolysis coupled with physico-chemical process based biomass fuels, the power densities of the cell were very similar to those values when using standard mixed glucose/xylose solutions (Table 1).
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Fig. 2. (A) CVs of the laccase/MWNTs/GCE biocathode in 0.2 M phosphate–citric acid buffer (pH 4.5) (a) without ABTS under N2-saturated atmosphere, (b) in the presence of 0.5 mM ABTS under N2-saturated and (c) in the presence of 0.5 mM ABTS under oxygen-saturated atmosphere. Scan rate, 20 mV s1. (B) Polarization curves of the laccase/MWNTs/GCE biocathode in 0.2 M phosphate–citric acid buffer (pH 4.5) (a) in the presence of 0.5 mM ABTS under N2-saturated atmosphere, (b) in the presence of 0.5 mM ABTS under ambient air, and (c) in the presence of 0.5 mM ABTS under oxygen-saturated atmosphere. Scan rate, 1 mV s1.
3.5. Stability of the BFC As the energy device, the lifetime is of great importance. Enzymatic biofuel cells suffer from an inevitable disadvantage for long-term operation, because of the loss of enzyme activity. To test the long-term stability of the assembled BFC, the cell was recorded continuously in as-prepared degradation products solution containing 10 mM NAD+ under ambient air. The power density increased during the first 3 h recording, after that, it began to decrease gradually (Fig. S2, Supplementary materials). Similar phenomena were observed in previous report (Yan et al., 2006). The exact reason for this is unclear for the moment. It is probably originating from the accumulation of the concentration of NADH during the beginning process. After 10 h operation, it still remained 80% its original maximal power (Fig. S2), indicating a favorably stable power output process, which may contribute to the improved stability of the mutant XDH and mutant GDH displayed on bacterial surface (Liang et al., 2013). 3.6. Faraday efficiency Faraday efficiency is an important parameter of a BFC, which is defined as the ratio of coulombs (amperes seconds) transferred from the substrate to the anode, to the theoretical maximum coulombs produced provided that all of the substrate in the cell
Fig. 3. (A) Dependence of the power density on the cell voltage: (a), xylose/O2 BFC, the anolyte was 0.1 M PBS buffer (pH 7.4) containing 15 mM xylose, 10 mM NAD+, 0.5 mM MB and XDH-bacteria; (b), glucose/O2 BFC, the anolyte was 0.1 M PBS buffer (pH 7.4) containing 25 mM glucose, 10 mM NAD+, 0.5 mM MB and GDHbacteria. (B) Dependence of the power density on the sugar fuel concentration: (a), xylose/O2 BFC; (b), glucose/O2 BFC.
Table 1 The power density using different degradation products of lignocellulose as fuels. Lignocellulose
Sugar conc. in the degradation products
Power density (lW cm2)c,d
Power density (lW cm2)d,e
Corn cob 1#a
Xylose (8.3 mM), glucose (19.7 mM) Xylose (8.6 mM), glucose (21.3 mM)
45.1 ± 3.8
48.5 ± 3.3
53.2 ± 1.9
52.4 ± 4.1
Corn stalk 2#b
a Corn cob 1#, the degradation products of corn cob, first treated with enzymatic hydrolysis and followed by physico-chemical process. b Corn stalk 2#, the degradation products of corn stalk, first treated with enzymatic hydrolysis, and followed by physico-chemical process. c The two-compartment degradation products of lignocellulose/O2 BFCs were assembled. The anolyte: 0.1 M PBS buffer (pH 7.4) containing 10 mM NAD+, 0.5 mM MB and XDH-bacteria and GDH-bacteria and lignocellulose degradation products. d The power density is expressed as the mean ± standard deviation for three separate experiments. e The two-compartment was the same as that of degradation products of lignocellulose/O2 BFCs. The anolyte: 0.1 M PBS buffer (pH 7.4) containing 10 mM NAD+, 0.5 mM MB and XDH-bacteria and GDH-bacteria with the mixture of standard xylose and glucose as fuels in the same concentration as in the degradation products.
is oxidized (Osman et al., 2011). This power production was accompanied by the consumption of diluted lignocellulose degradation products. The Faraday efficiency in this work was about 41% (Fig. 4), which was higher than that value of 30% for
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References
Fig. 4. Profiles for current generation (a) and cumulative Faraday efficiency of the assembled BFC (b).
glucose/O2 biofuel cell (Velasquez-Orta et al., 2011). Generally, two-compartment may lead to higher power outputs with low faraday efficiencies (Osman et al., 2011). 4. Conclusions The degradation products of lignocellulosic biomass/O2 BFC in two-compartment was fabricated for the first time, which exhibited a maximum power density of 53 lW cm2 with degradation products of corn stalk as fuel. Moreover, the assembled BFC exhibited a high open-circuit potential of 0.80 V, acceptable stability and energy conversion efficiency. It is envisioned that the assembled BFC is promising to use lignocellulosic biomass as source energy. Acknowledgements This work was financially supported by National Natural Science Foundation of China (Nos. 91227116, 21275152, and 21475144), the Postdoctoral Science Foundation of China (No. 2014M560586), Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences (CASKLB201505) and the Qingdao Institute of Bioenergy and Bioprocess Technology Director Innovation Foundation for Young Scientists (Y37203210S). 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.2015.05. 090.
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