Membraneless ethanol,O 2 enzymatic biofuel cell based on laccase and ADH/NAD + bioelectrodes

: This work describes EtOH,O 2 membraneless enzymatic biofuel cells (EtOH,O 2 M less EBFCs) that employ laccase-based biocathodes and ADH/NAD + bioanode. Laccase biocathodes were prepared by immobilizing a polypyrrole film containing different redox mediators (ruthenium and osmium complexes). The bioanode for EtOH,O 2 MlessEBFCs was fabricated by immobilizing multiwalled carbon nanotubes, NAD + -dependent alcohol dehydrogenase enzyme (ADH), poly-methylene green, and poly(amidoamine) (PAMAM) dendrimer onto a carbon cloth platform. Maximum power density and current density were 21.0 ± 0.2  W cm -2 and 0.15 ± 0.07 mA cm -2 , respectively, in PBS (pH 6.5). Lifetime tests conducted for EtOH,O 2 M less EBFCs showed promising perspectives for their future application in miniaturized devices.


Introduction
Biofuel cells (BFCs) employ enzymes or microorganisms as catalysts to convert chemical energy into electric energy.BFCs can operate under milder temperatures (20-40 °C) and physiological pH, so they could be a strategy to replace traditional batteries that need large amount of hazardous metallic catalysts in small devices.Moreover, BFCs could be employed to produce energy from various fuel sources because enzymes can selectively catalyze different fuels 1,2 .Enzymes have high specific selectivity, which could dismiss the need for a membrane 3 .The first report of a membraneless biofuel cell (MlessBFC) dates from 1997, when a single compartment cell was used to oxidize organic compounds (sugars or alcohols) while simultaneously reducing molecular oxygen (O2) at the biocathode 4 .
To prepare MlessBFCs successfully, enzyme immobilization is a key step to obtain a stable longlasting device, improving electron transfer kinetics, and increasing power densities (PDs).In this context, researchers have sought to enhance enzymatic system robustness and activity-an enzymatic system must be able to survive pH, temperature, and reaction medium changes.This is not a simple task when one deals with biomolecules, but growing interest in this area has advanced knowledge in the field.A 20-day lifetime has been reported for a membraneless ethanol, oxygen enzymatic biofuel cell (EtOH,O2 MlessEBFC) based on alcohol dehydrogenase (ADH) and bilirubin oxidase (BOD) as bioelectrodes 5 .Over the years, numerous architecture designs for MlessEBFCs have been developed in order to achieve higher PD values 6,7 .For instance, Deng and co-workers 8 produced ABSTRACT: This work describes EtOH,O2 membraneless enzymatic biofuel cells (EtOH,O2 MlessEBFCs) that employ laccase-based biocathodes and ADH/NAD + bioanode.Laccase biocathodes were prepared by immobilizing a polypyrrole film containing different redox mediators (ruthenium and osmium complexes).The bioanode for EtOH,O2 MlessEBFCs was fabricated by immobilizing multiwalled carbon nanotubes, NAD + -dependent alcohol dehydrogenase enzyme (ADH), polymethylene green, and poly(amidoamine) (PAMAM) dendrimer onto a carbon cloth platform.Maximum power density and current density were 21.0 ± 0.2 W cm -2 and 0.15 ± 0.07 mA cm -2 , respectively, in PBS (pH 6.5).Lifetime tests conducted for EtOH,O2 MlessEBFCs showed promising perspectives for their future application in miniaturized devices.
To increase PD, carbon-based materials, such as multiwalled carbon nanotubes (MWCNTs), have been successfully investigated 9 .With respect to electrochemical performance, MWCNTs are claimed to be more efficient than single-walled carbon nanotubes (SWCNTs).Indeed, MWCNTs have greater surface area and wider potential range, provide many active sites for biomolecule immobilization (which promotes faster electron transfer reactions along the tube axis), and display prominent charge transortation features 10,11 .
Immobilization aiming at protein microencapsulation has currently gained researchers' attention.In this immobilization mode, intrinsically conductive polymers and dendrimers are employed as imprisonment arrays so that enzymes are physically entrapped in membrane pores or anchored onto the electrode surface.Intrinsically conductive polymers are compounds that can carry electric current without incorporating conductive charges.Also known as conjugated polymers, their electrical, optical, magnetic, and electronic properties resemble the properties of metals and/or semiconductors.Here, we highlight the use of polypyrrole (polyPYR), which is highly chemically and environmentally stable, biocompatible, and biodegradable.This porous polymer has been widely applied in batteries, sensors, and anti-corrosion protective agents, among others.Several methodologies can be employed to obtain polyPYR layers, and use of this polymer, modified or not, has been often reported [12][13][14] .Our research group prepared enzymatic biocathode and bioanode for biofuel cells 15,16 , and an example of MlessEBFCs application can be found elsewhere 17 .PolyPYR and MWCNT matrixes have been employed to prepare glucose,O2 EBFCs based on glucose oxidase (GOx) and pyrroloquinoline quinone (PQQ) redox mediator absorbed on MWCNTs and polyPYR as a MWCNTs-GOx-PQQ-polyPYR bioanode 17 .PD of 1.1 W mm -2 was achieved at non-compartmentalized BFCs at a cell voltage of 0.167 V in PBS (pH 7.4) for 10 mM glucose (as fuel), and PD of 0.69 W mm -2 was obtained at cell voltage of 0.151 V in human serum containing 5 mmol L -1 glucose (37 °C) 17 .
PAMAM dendrimer is another promising polymer belonging to the class of branched monodisperse polymers 18,19 .PAMAM has widely uniform structure, low molecular weight, highly functionalized surface and high degree of porosity 19 .
We report the construction of a single-chamber EtOH,O2 biofuel cell to harvest energy from ethanol.Strategies to enhance ET between enzymes and electroactive surfaces include orientation and immobilization of the enzymes and electron mediation.For this laccase-based biocathode metallic redox complexes (Os and Ru) was entrapped in a polyPYR film as redox mediators and the ADH/NAD + bioanode employed polymethylene green layer as mediator.We also investigate the activity of the membraneless biofuel for a long period (11 months) in other to show their stability.
Multiwalled carbon nanotubes (MWCNTs) were acquired from Cheap Tubes Inc. (diameter of 8.0 nm, length of 10 to 30 m, and > 95% purity).All solutions were prepared with high-purity water from a Millipore Milli-Q system.Solution pH was measured with a pH electrode coupled to a Qualxtron model 8010 pHmeter.
pH influence on enzymatic kinetics was determined by assaying laccase and ADH activities at various pH values ranging between 3.5 and 10.To this end, the following 0.1 mol L −1 buffer solutions were employed: acetate buffer (NaAc/HAc) for pH 3.5-5, phosphate buffer (NaH2PO4/Na2HPO4) for pH 6-7, and tris(hydroxymethyl)aminomethane-HCl (Tris+) buffer for pH 8-9.Reaction was initiated by adding substrate to the immobilized protein, depending on the study that was being performed.

Biofuel cell tests
Power density measurements were accomplished in an EtOH,O2 MlessEBFC described in the Instrumentation Section.First, the EtOH,O2 MlessEBFC open circuit voltage (OCV) was measured at least 1 h before the cell test.After that, polarization curves at a scan rate of 1 mV s -1 were registered in triplicate.PD values for all MlessEBFCs were obtained by multiplying cell voltage (Ecell) by current density (Jcell) (PD = Ecell × Jcell).

pH effect on semi-MlessEBFC
To obtain maximum MlessEBFC performance, it is important to investigate bioelectrode enzymatic behavior as a function of pH because hydrogen ion concentration affects enzymatic activity: enzyme spatial conformation depends on pH values and on the presence of protonated/deprotonated groups in the enzyme catalytic site, which can modify the enzyme tertiary structure.pH also influences intrinsic/extrinsic electron transfer reaction strongly.The individual pH behavior of the immobilized enzymes employed here has previously been investigated in detail 15,20 .The optimum pH range for ADH/NAD + bioanode is between 7.0 and 8.0, achieved by employing PBS as buffer 24 .Laccase works best in more acidic medium (pH 4.5), in ABS buffer solution 15 .Therefore, besides operating in different pH ranges, bioanode and biocathode also use distinct buffer solutions.
Direct correlation between enzymatic kinetics and pH is important to obtain maximum bioelectrode performance.Nevertheless, in a MlessEBFC both bioelectrodes seldom operate at their optimum pH.To find the best pH for MlessEBFC operation, individual pH curves of each enzyme were plotted together (Fig. 2).On the basis of Fig. 2, pH curves intersect at pH 6.5, which was further employed with MlessEBFCs.Even though this pH value is close to physiological conditions, other complications may arise and diminish EtOH,O2 MlessEBFC PD and OCV values as compared to separated biofuel cells.Other factors may also be associated with this behavior such as problems with the enzyme-mediator-electrode electron transfer enzyme, which hinders the redox process underlying EtOH oxidation by ADH and O2 reduction to H2O by multicopper oxidase enzymes (laccase).

EtOH,O2 membraneless biofuel cell: reaction medium influence (ABS or PBS).
Eliminating proton exchange membrane (PEM) has several advantages.During the reaction process, PEM is subjected to membrane channel obstruction by ions present in the supporting electrolyte, which dries or floods membrane parts, and fuel crossover.To minimize the aforementioned problems, one strategy is to remove the membrane when biocatalyst specificity can be maintained at MlessEBFC.However, each bioelectrode must be evaluated for its electron transfer activity and enzymatic selectivity.EtOH,O2 MlessEBFC performance was assessed by analyzing OCV and power density curves obtained from polarization curves (results not shown).To investigate how PBS and ABS buffers influenced MlessEBFC activity, EtOH,O2 MlessEBFC performance was measured at pH 6.5 in both buffers (ABS and PBS; buffer concentration = 200 mmol L -1 ).This "precaution" was necessary because PEM removal resulted in each enzyme facing a medium that was different from its ideal operation condition (ABS (pH 4.5) for laccase and PBS (pH 7.4) for ADH).

ABTS influence on EtOH,O2 MlessBFCs
ABTS is one of the most common oxygen reduction mediators when laccase is employed in EBFCs.Figure 4 illustrates how ABTS influences EtOH,O2 MlessEBFC performance in PBS medium (pH 6.5) for both anode configurations investigated here: polyPYR-Os-laccase and polyPYR-Rulaccase.Homogeneous ABTS introduction diminishes Ru-mediated cathode cell PD by over 80% as compared to Os-mediated cathode cell.Indeed, PD decreased from 15.3 ± 0.2 W cm -2 to 2.7 ± 0.3 W cm -2 just by changing the Os complex to the Ru complex.Also, when results obtained in the absence (Fig. 3A) and in the presence (Fig. 4) of ABTS are compared for Os-complex in PBS, PD and Jcell(max) was approximately 27.5 and 23.8%, respectively.This decrease could be explained by competition between ABTS and mediators incorporated into the polyPYR matrix and the enzymatic redox sites.The best results were achieved for EtOH,O2 MlessEBFCs based on the MWCNT-ADH,polyPYR-Os-laccase system.These results agreed with literature data 25 claiming that [Os(bpy)2Cl2] can bind to laccase copper hydrophobic T1 active site and establish a strong electrostatic interaction, which entraps the Oscomplex in polyPYR, shifts the polyPYR-Os oxidation potential (Eoxi), and facilitates electron transfer.Our results showed that polyPYR-Rulaccase did not interact in the same way as the Osmediator.
Table 1 summarizes all experimental parameters obtained for EtOH,O2 MlessEBFCs as a function of the different redox mediators entrapped in polyPYR, in the absence or presence of 1 mmol L -1 ABTS.On the basis of the results above (Table 1), the best EtOH,O2 MlessEBFCs was MWCNTs-ADH,polyPYR-Os-laccase in 200 mmol L -1 PBS (pH 6.5) in the absence of ABTS. Figure 5 illustrates the selected operation system.We have investigated and reported half-cell data for these electrodes configuration before 15,21 .For the biocathode half-cell 15 a gas diffusion membrane (ELAT) consisting of 40% metal in C (Pt0.66Ru0.34,E-TEK commercial mixture) hot pressed in a Nafion NRE-212 membrane was employed as the anode.This configuration furnished at least five times higher power densities values than the value reported for the membraneless fuel cell.For the half-bioanode 21 Pt was used as cathode, also separated by a Nafion membrane.This configuration furnished power density values as high as 0.25 mW cm -2 .Table 1 shows that the results for EtOH,O2 MlessEBFCs are much lower than the separated compartment cell indicating that besides pH effect must be a mutual influence of the fuel and O2 in the performance of the enzymatic systems.Nevertheless, despite differences with respect to enzyme immobilization method, the values measured herein are in the same order of magnitude (W cm -2 ) with some data reported in the literature data 5,8,27,28 .Considering the high efficiency in ethanol/acetaldehyde conversion and concomitant O2 reduction to H2O demonstrated by enzymatic systems future application of biofuel cells in miniaturized systems must be solved preparing microfluidic devices that operate with streams of liquid electrolytes 29 .In this configuration it is possible to operate with different electrolytes for anodic and cathodic compartments without any problem.This is our future goal to increase the power density.
Table 2 lists the MWCNTs-ADH,polyPYR-Oslaccase cell storage lifetime under optimum conditions (200 mmol L -1 PBS (pH 6.5), 1.9 mmol L -1 NAD + , and 100 mmol L -1 EtOH).After five months, PD and Jcell(max) decreased by approximately 38% (13 ± 4) W cm -2 and 0.09 ± 0.02 mA cm -2 , respectively) as compared to freshly prepared electrodes.These values dropped slowly and reached 62% and 48% (8 ± 2 W cm -2 and 0.09 ± 0.02 mA cm -2 , respectively) of the initial values.These results attested that immobilization of the enzymes employed here provided a relatively stable medium for long-term storage tests.This result may be important to apply these devices in new types of nanofluid cells to enhance the power harvest from the ethanol molecule 30 .

Conclusions
Bioelectrodes containing the enzymes ADH and laccase and different redox mediators (Os or Ru) entrapped in polyPYR films or PAMAM dendrimer were tested.MWCNTs-ADH,polyPYR-Os-laccase employing PBS (pH 6.5) in the absence of ABTS performed the best.PD and Jcell(max) remained around 21.0 ± 0.2 W cm -2 and 0.15 ± 0.07 mA cm - 2 for freshly prepared electrodes.Electrodes retain 38% of their activity after storage for five months storage in a refrigerator.The prepared EtOH,O2 MlessEBFC generated power densities values comparable with literature data as well as considerable lifetime stability.Therefore, the results presented here for EtOH,O2 MlessEBFCs are promising and may be employed in microfluidic devises to enhance the activity of the system.

Table 1 .
Parameters obtained for different EtOH/O2 MlessEBFCs.Average and standard deviation for combinatorial analysis, in triplicate, for a set of three biocathodes and four bioanodes. *