Bioelectrochemistry (v.106, #PA)

Table of Contents (vii-viii).

Biological fuel cells: Divergence of opinion by Sergey Shleev; Alain Bergel; Lo Gorton (1-2).

Dynamic and steady state 1-D model of mediated electron transfer in a porous enzymatic electrode by T.Q.N. Do; M. Varničić; R.J. Flassig; T. Vidaković-Koch; K. Sundmacher (3-13).
A 1-D mathematical model of a porous enzymatic electrode exhibiting the mediated electron transfer (MET) mechanism has been developed. As a model system, glucose oxidation catalyzed by immobilized glucose oxidase (GOx) in the presence of a co-immobilized tetrathiafulvalene (TTF) mediator in the porous electrode matrix has been selected. The balance equations for potential fields in the electron- and ion-conducting phases as well as concentration field have been formulated, solved numerically and validated experimentally under steady state conditions. The relevant kinetic parameters of the lumped reaction kinetics have been obtained by global optimization. The confidence intervals (CIs) of each parameter have been extracted from the respective likelihood. The parameter study has shown that the parameters related to mediator consumption/regeneration steps can be responsible for the shift of the reaction onset potential. Additionally, the model has shown that diffusion of the oxidized mediator out of the catalyst layer (CL) plays a significant role only at more positive potentials and low glucose concentrations. Only concentration profiles in different layers influence the electrode performance while other state fields like potential distributions in different phases have no impact on the performance. The concentration profiles reveal that all electrodes work through; the observed limiting currents are diffusion–reaction limiting. The normalized electrode activity decreases with an increase of enzyme loading. According to the model, the reason for this observation is glucose depletion along the CL at higher enzyme loadings. Comparison with experiments advices a decrease of enzyme utilization at higher enzyme loadings.Display Omitted
Keywords: Glucose oxidase (GOx); Tetrathiafulvalene (TTF); Mediated electron transfer (MET); Porous enzymatic electrode model; Kinetic parameters;

Wired enzymes in mesoporous materials: A benchmark for fabricating biofuel cells by Paolo N. Catalano; Alejandro Wolosiuk; Galo J.A.A. Soler-Illia; Martín G. Bellino (14-21).
Evolution of fuel cells using metallic inorganic catalysts has led to the development of biofuel cells with potential applications in implantable devices. However, the main disadvantages in real world applications of enzymatic biofuel cells are short lifetime and low power density. Many efforts have been devoted to immobilize redox enzymes on surfaces to allow efficient electrical communication with electrodes and to provide an adequate habitat for biochemical activity. In this context, nanocavities of mesoporous materials offer a tailored environment for protein immobilization. Mesostructured platforms with high surface area and stability have been developed to enhance mass transport, charge transfer from biocatalysts to electrodes and enzyme stability, leading to biofuel cells with improved power density (up to 602 μW cm− 2 at physiological conditions) and overall performance (high stability after 30 h of continuous operation and after 10 days of storage). This review discusses recent developments using mesoporous materials as novel platforms for effective electronic charge transfer in the context of current and emerging technologies in enzymatic fuel cell research, emphasizing their practical implications and potential improvements leading to a major impact on medical science and portable electronics.
Keywords: Mesoporous materials; Enzymatic biofuel cells; Enzyme wiring;

Coupling of an enzymatic biofuel cell to an electrochemical cell for self-powered glucose sensing with optical readout by Piyanut Pinyou; Felipe Conzuelo; Kirill Sliozberg; Jeevanthi Vivekananthan; Andrea Contin; Sascha Pöller; Nicolas Plumeré; Wolfgang Schuhmann (22-27).
A miniaturized biofuel cell (BFC) is powering an electrolyser invoking a glucose concentration dependent formation of a dye which can be determined spectrophotometrically. This strategy enables instrument free analyte detection using the analyte-dependent BFC current for triggering an optical read-out system. A screen-printed electrode (SPE) was used for the immobilization of the enzymes glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) for the biocatalytic oxidation of glucose and reduction of molecular oxygen, respectively. The miniaturized BFC was switched-on using small sample volumes (ca. 60 μL) leading to an open-circuit voltage of 567 mV and a maximal power density of (6.8 ± 0.6) μW cm− 2. The BFC power was proportional to the glucose concentration in a range from 0.1 to 1.0 mM (R 2  = 0.991). In order to verify the potential instrument-free analyte detection the BFC was directly connected to an electrochemical cell comprised of an optically-transparent SPE modified with methylene green (MG). The reduction of the electrochromic reporter compound invoked by the voltage and current flow applied by the BFC let to MG discoloration, thus allowing the detection of glucose.Display Omitted
Keywords: Biofuel cell; Screen-printed electrode; Instrument-free analysis; Glucose determination; Self-powered device; Optical read-out;

A biofuel cell composed of catalytic electrodes made of “buckypaper” modified with PQQ-dependent glucose dehydrogenase and FAD-dependent fructose dehydrogenase on the anode and with laccase on the cathode was used to activate a wireless information transmission system. The cathode/anode pair was implanted in orange pulp extracting power from its content (glucose and fructose in the juice). The open circuit voltage, Voc, short circuit current density, jsc, and maximum power produced by the biofuel cell, Pmax, were found as ca. 0.6 V, ca. 0.33 mA·cm− 2 and 670 μW, respectively. The voltage produced by the biofuel cell was amplified with an energy harvesting circuit and applied to a wireless transmitter. The present study continues the research line where different implantable biofuel cells are used for the activation of electronic devices. The study emphasizes the biosensor and environmental monitoring applications of implantable biofuel cells harvesting power from natural sources, rather than their biomedical use.Display Omitted
Keywords: Implantable biofuel cell; Wireless transmission; Orange; Glucose dehydrogenase; Fructose dehydrogenase; Laccase;

Biosupercapacitors for powering oxygen sensing devices by Michal Kizling; Sylwia Draminska; Krzysztof Stolarczyk; Petter Tammela; Zhaohui Wang; Leif Nyholm; Renata Bilewicz (34-40).
A biofuel cell comprising electrodes based on supercapacitive materials — carbon nanotubes and nanocellulose/polypyrrole composite was utilized to power an oxygen biosensor. Laccase Trametes versicolor, immobilized on naphthylated multi walled carbon nanotubes, and fructose dehydrogenase, adsorbed on a porous polypyrrole matrix, were used as the cathode and anode bioelectrocatalysts, respectively. The nanomaterials employed as the supports for the enzymes increased the surface area of the electrodes and provide direct contact with the active sites of the enzymes. The anode modified with the conducting polymer layer exhibited significant pseudocapacitive properties providing superior performance also in the high energy mode, e.g., when switching on/off the powered device. Three air–fructose biofuel cells connected in a series converted chemical energy into electrical giving 2 mW power and open circuit potential of 2 V. The biofuel cell system was tested under various externally applied resistances and used as a powering unit for a laboratory designed two-electrode minipotentiostat and a laccase based sensor for oxygen sensing. Best results in terms of long time measurement of oxygen levels were obtained in the pulse mode − 45 s for measurement and 15 min for self-recharging of the powering unit.
Keywords: Supercapacitor; Biofuel cell; Laccase; Fructose dehydrogenase; Polypyrrole; Nanocellulose; Oxygen biosensor;

This study reports a design of experiment methodology to investigate and improve the performance of glucose oxidizing enzyme electrodes. Enzyme electrodes were constructed by co-immobilization of amine-containing osmium redox complexes, multiwalled carbon nanotubes and glucose oxidase in a carboxymethyldextran matrix at graphite electrode surfaces to provide a 3-dimensional matrix for electrocatalytic oxidation of glucose. Optimization of the amount of the enzyme electrode components to produce the highest current density under pseudo-physiological conditions of 5 mM glucose in saline buffer at 37 °C was performed using response surface methodology. A statistical analysis showed that the proposed model had a good fit with the experimental results. From the validated model, the addition of multiwalled carbon nanotubes and carboxymethyldextran components was identified as major contributing factors to the improved performance. Based on the optimized amount of components, enzyme electrodes display current densities of 1.2 ± 0.1 mA cm− 2 and 5.2 ± 0.2 mA cm− 2 at 0.2 V vs. Ag/AgCl in buffer containing 5 mM and 100 mM glucose, respectively, largely consistent with the predicted values. This demonstrates that use of a design of experiment approach can be applied effectively and efficiently to improve the performance of enzyme electrodes as anodes for biofuel cell device development.
Keywords: Biofuel cell; Osmium mediator; Glucose oxidation; Design of experiment;

Hydrogen bioelectrooxidation on gold nanoparticle-based electrodes modified by Aquifex aeolicus hydrogenase: Application to hydrogen/oxygen enzymatic biofuel cells by Karen Monsalve; Magali Roger; Cristina Gutierrez-Sanchez; Marianne Ilbert; Serge Nitsche; Deborah Byrne-Kodjabachian; Valérie Marchi; Elisabeth Lojou (47-55).
For the first time, gold nanoparticle-based electrodes have been used as platforms for efficient immobilization of the [NiFe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. AuNPs were characterized by electronic microscopy, dynamic light scattering and UV–Vis spectroscopy. Two sizes around 20.0 ± 5.3 nm and 37.2 ± 4.3 nm nm were synthesized. After thiol-based functionalization, the AuNPs were proved to allow direct H2 oxidation over a large range of temperatures. A high current density up to 1.85 ± 0.15 mA·cm− 2 was reached at the smallest AuNPs, which is 170 times higher than the one recorded at the bare gold electrode. The catalytic current was especially studied as a function of the AuNP size and amount, and procedure for deposition. A synergetic effect between the AuNP porous deposit and the increase surface area was shown. Compared to previously used nanomaterials such as carbon nanofibers, the covalent grafting of the enzyme on the thiol-modified gold nanoparticles was shown to enhance the stability of the hydrogenase. This bioanode was finally coupled to a biocathode where BOD from Myrothecium verrucaria was immobilized on AuNP-based film. The performance of the so-mounted H2/O2 biofuel cell was evaluated, and a power density of 0.25 mW·cm− 2 was recorded.
Keywords: Gold nanoparticles; Hydrogenase; Bilirubin oxidase; Direct electron transfer; Enzymatic H2/O2 biofuel cell;

Employing FAD-dependent glucose dehydrogenase within a glucose/oxygen enzymatic fuel cell operating in human serum by Ross D. Milton; Koun Lim; David P. Hickey; Shelley D. Minteer (56-63).
Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) is emerging as an oxygen-insensitive alternative to glucose oxidase (GOx) as the biocatalyst for bioelectrodes and bioanodes in glucose sensing and glucose enzymatic fuel cells (EFCs). Glucose EFCs, which utilize oxygen as the oxidant and final electron acceptor, have the added benefit of being able to be implanted within living hosts. These can then produce electrical energy from physiological glucose concentrations and power internal or external devices. EFCs were prepared with FAD-GDH and bilirubin oxidase (BOx) to evaluate the suitability of FAD-GDH within an implantable setting. Maximum current and power densities of 186.6 ± 7.1 μA cm− 2 and 39.5 ± 1.3 μW cm− 2 were observed when operating in human serum at 21 °C, which increased to 285.7 ± 31.3 μA cm− 2 and 57.5 ± 5.4 μW cm− 2 at 37 °C. Although good stability was observed with continual near-optimal operation of the EFCs in human serum at 21 °C for 24 h, device failure was observed between 13–14 h when continually operated at 37 °C.
Keywords: FAD-dependent glucose dehydrogenase; Bilirubin oxidase; Serum; Glucose; Oxygen;

Analysis of bio-anode performance through electrochemical impedance spectroscopy by Annemiek ter Heijne; Olivier Schaetzle; Sixto Gimenez; Lucia Navarro; Bert Hamelers; Francisco Fabregat-Santiago (64-72).
In this paper we studied the performance of bioanodes under different experimental conditions using polarization curves and impedance spectroscopy. We have identified that the large capacitances of up to 1 mF·cm− 2 for graphite anodes have their origin in the nature of the carbonaceous electrode, rather than the microbial culture.In some cases, the separate contributions of charge transfer and diffusion resistance were clearly visible, while in other cases their contribution was masked by the high capacitance of 1 mF·cm− 2. The impedance data were analyzed using the basic Randles model to analyze ohmic, charge transfer and diffusion resistances. Increasing buffer concentration from 0 to 50 mM and increasing pH from 6 to 8 resulted in decreased charge transfer and diffusion resistances; lowest values being 144 Ω·cm2 and 34 Ω·cm2, respectively. At acetate concentrations below 1 mM, current generation was limited by acetate. We show a linear relationship between inverse charge transfer resistance at potentials close to open circuit and saturation (maximum) current, associated to the Butler–Volmer relationship that needs further exploration.
Keywords: Electrochemical impedance spectroscopy; Internal resistance; Microbial fuel cell; Charge transfer; Diffusion;

One-year stability for a glucose/oxygen biofuel cell combined with pH reactivation of the laccase/carbon nanotube biocathode by Bertrand Reuillard; Caroline Abreu; Noémie Lalaoui; Alan Le Goff; Michael Holzinger; Olivier Ondel; Francois Buret; Serge Cosnier (73-76).
This study reports a mixed operational/storage stability of a MWCNT-based glucose biofuel cell (GBFC) over one year. The latter was examined by performing a one hour discharge every day during one month followed by several discharges over a period of 11 months. Under continuous discharge in physiological conditions (5 mM glucose, 37°, pH 7), the GBFC exhibits a 25% power decrease after 1 h of operation. This decrease is mainly due to the deactivation of laccase biocathodes at neutral pH. Nevertheless, the biocathodes can be reversibly reactivated via storage in phosphate buffer (pH 5). Under these conditions, the GBFC finally exhibits 22% of its initial maximum power density after one year at intermittent reactivation/discharge cycles. Although both GBFC electrodes can exhibit one year stability, short-term experiments show that biocathodes are limited by hydroxide inhibition while long-term experiments indicate that bioanodes are likely limited by the stability of the GOx itself. While most of the GBFCs in the literature present stability in the range of several weeks, these results demonstrate the viability of a GBFC for industrial applications in a long period of time.
Keywords: Glucose biofuel cell; Laccase; Carbon nanotubes; Glucose oxidation; Direct electron transfer;

Oxidation of laccase for improved cathode biofuel cell performances by Meihui Zheng; Sophie Griveau; Christine Dupont-Gillain; Michel J. Genet; Claude Jolivalt (77-87).
Graphite rods were modified by substituted aryldiazonium salts allowing subsequent laccase immobilisation and direct electron transfer at the cathode. Two covalent enzyme immobilisation methods were performed with carboxy and amino substituted grafted groups, either via the formation of an amide bond or a Schiff base between the glycosidic groups of the enzyme and the amino groups on the electrode surface, respectively. Laccase adsorption efficiency was consistently compared to the covalent attachment method on the same carbon surface, showing that the latter method led to a higher immobilisation yield when the electrode surface was functionalised with carboxylic groups, as shown from both laccase activity measurement towards an organic reducing substrate, ABTS, and quantitative XPS analysis. Both analytical methods led to similar laccase surface coverage estimations. From activity measurements, when laccase was covalently immobilised on the electrode functionalised with carboxylic groups, the surface coverage was found to be 43 ± 2% whereas it was only 10 ± 3% when laccase was adsorbed. Biocatalysed dioxygen reduction current was also higher in the case of covalent immobilisation. For the first time, oxidised laccase performances were compared to unmodified laccase, showing significant improved efficiency when using oxidised laccase: the current obtained with oxidised laccase was 141 ± 37 μA cm− 2 compared to 28 ± 6 μA cm− 2 for unmodified laccase after covalent immobilisation of the enzyme on a graphite electrode functionalised with carboxylic groups.
Keywords: Biofuel cell; Direct electron transfer; Oxidised laccase; Biocathode; Diazonium salt; Biocatalytic dioxygen reduction; XPS;

All ecosystems potentially host electrogenic bacteria by Nicolas Chabert; Oulfat Amin Ali; Wafa Achouak (88-96).
Instead of requiring metal catalysts, MFCs utilize bacteria that oxidize organic matter and either transfer electrons to the anode or take electrons from the cathode. These devices are thus based on a wide microbial diversity that can convert a large array of organic matter components into sustainable and renewable energy. A wide variety of explored environments were found to host electrogenic bacteria, including extreme environments. In the present review, we describe how different ecosystems host electrogenic bacteria, as well as the physicochemical, electrochemical and biological parameters that control the currents from MFCs. We also report how using new molecular techniques allowed characterization of electrochemical biofilms and identification of potentially new electrogenic species. Finally we discuss these findings in the context of future research directions.
Keywords: Biofilm composition; Exocellular electron transfer; Microbial fuel cells; Inocula source; Molecular tools; Imagery;

Electrochemical characterization of microbial bioanodes formed on a collector/electrode system in a highly saline electrolyte by Raphaël Rousseau; Mickaël Rimboud; Marie-Line Délia; Alain Bergel; Régine Basséguy (97-104).
Bioanodes were formed with electrodes made of carbon felt and equipped with a titanium electrical collector, as commonly used in microbial fuel cells. Electrochemical impedance spectroscopy (EIS) performed on the abiotic electrode system evidenced two time constants, one corresponding to the “collector/carbon felt” contact, the other to the “carbon felt/solution” interface. Such a two time constant system was characteristics of the two-material electrode, independent of biofilm presence. EIS was then performed during the bioanode formation around the constant applied potential of 0.1 V/SCE. The equivalent electrical model was similar to that of the abiotic system. Due to the high salinity of the electrolyte (45 g·L− 1 NaCl) the electrolyte resistance was always very low. The bioanode development induced kinetic heterogeneities that were taken into account by replacing the pure capacitance of the abiotic system by a constant phase element for the “carbon felt/solution” interface. The current increase from 0 to 20.6 A·m− 2 was correlated to the considerable decrease of the charge transfer resistance of the “carbon felt/solution” interface from 2.4 104 to 92 Ω·cm2. Finally, EIS implemented at 0.4 V/SCE showed that the limitation observed at high potential values was not related to mass transfer but to a biofilm-linked kinetics.
Keywords: Bioanode; Electrochemical impedance spectroscopy; Halotolerant; Limiting step; Microbial fuel cell;

The electrical parameters of nine freshwater sediment microbial fuel cells (SMFCs) were monitored for a period of over 20 months. The developed SMFCs, divided into three groups, were started up and continuously operated under different constant loads (100, 510 and 1100 Ω) for 2.5 months. At this stage of the experiment, the highest power density values, reaching 1.2 ± 0.2 mW/m2, were achieved by the SMFCs loaded with 510 Ω. The maximum power obtained at periodical polarization during the rest period, however, ranged between 26.2 ± 2.8 and 35.3 ± 2.8 mW/m2, strongly depending on the internal cell resistance. The statistical evaluation of data derived from the polarization curves shows that after 300 days of operation all examined SMFCs reached a steady-state and the system might be assumed as homoscedastic. The estimated values of standard and expanded uncertainties of the electric parameters indicate a high repeatability and reproducibility of the SMFCs' performance. Results obtained in subsequent discharge–recovery cycles reveal the opportunity for practical application of studied SMFCs as autonomous power sources.Display Omitted
Keywords: Sediment microbial fuel cells; Freshwater sediments; Statistics; Sustainability; Autonomous power generation;

Bio-electrochemical characterization of air-cathode microbial fuel cells with microporous polyethylene/silica membrane as separator by Nina Kircheva; Jonathan Outin; Gérard Perrier; Julien Ramousse; Gérard Merlin; Emilie Lyautey (115-124).
The aim of this work was to study the behavior over time of a separator made of a low-cost and non-selective microporous polyethylene membrane (RhinoHide®) in an air-cathode microbial fuel cell with a reticulated vitreous carbon foam bioanode. Performances of the microporous polyethylene membrane (RhinoHide®) were compared with Nafion®-117 as a cationic exchange membrane. A non-parametric test (Mann–Whitney) done on the different sets of coulombic or energy efficiency data showed no significant difference between the two types of tested membrane (p < 0.05). Volumetric power densities were ranging from 30 to 90 W·m− 3 of RVC foam for both membranes. Similar amounts of biomass were observed on both sides of the polyethylene membrane illustrating bacterial permeability of this type of separator. A monospecific denitrifying population on cathodic side of RhinoHide® membrane has been identified. Electrochemical impedance spectroscopy (EIS) was used at OCV conditions to characterize electrochemical behavior of MFCs by equivalent electrical circuit fitted on both Nyquist and Bode plots. Resistances and pseudo-capacitances from EIS analyses do not differ in such a way that the nature of the membrane could be considered as responsible.
Keywords: Electrochemical impedance spectroscopy; Reticulated carbon foam; Polyethylene membrane; Nonionic membrane;

Electrochemical and microbial monitoring of multi-generational electroactive biofilms formed from mangrove sediment by Caroline Rivalland; Sonia Madhkour; Paule Salvin; Florent Robert (125-132).
Electroactive biofilms were formed from French Guiana mangrove sediments for the analysis of bacterial communities' composition. The electrochemical monitoring of three biofilm generations revealed that the bacterial selection occurring at the anode, supposedly leading microbial electrochemical systems (MESs) to be more efficient, was not the only parameter to be taken into account so as to get the best electrical performance (maximum current density). Indeed, first biofilm generations produced a stable current density reaching about 18 A/m2 while second and third generations produced current densities of about 10 A/m2. MES bacterial consortia were characterized thanks to molecular biology techniques: DGGE and MiSeq® sequencing (Illumina®). High-throughput sequencing data statistical analysis confirmed preliminary DGGE data analysis, showing strong similarities between electroactive biofilms of second and third generations, but also revealing both selection and stabilization of the biofilms.
Keywords: Dissimilarity; High-throughput sequencing; Microbial diversity; MES; Syntrophism;

Successive bioanode regenerations to maintain efficient current production from biowaste by A. Bridier; E. Desmond-Le Quemener; C. Bureau; P. Champigneux; L. Renvoise; J.-M. Audic; E. Blanchet; A. Bergel; T. Bouchez (133-140).
The long-term operation of efficient bioanodes supplied with waste-derived organics is a key challenge for using bioelectrochemical systems as a waste valorization technology. Here, we describe a simple procedure that allowed maintaining highly efficient bioanodes supplied with biowaste. Current densities up to 14.8 A/m2 were obtained with more than 40% of the electrons introduced as biowaste being recovered in the electrical circuit. Three fed-batch reactors were started at different biowaste loading rates. A decline of coulombic efficiencies between 22 and 31% was recorded depending on the reactor over the first 3 weeks of operation. A renewal procedure of the anode was thereafter implemented, which led to a recovery of initial performances. The second and the third renewal, allowed maintaining stable high level performances with coulombic efficiency of approximately 40% over at least 3 weeks. Electroactive biofilm dynamics were monitored using 16S rRNA-gene amplicon sequencing. Retrieved sequences were dominated by Geobacter sulfurreducens-related reads (37% of total sequences), which proportion however varied along the experiment. Interestingly, sequences affiliated to various Bacteroidetes groups were also abundant, suggesting an adaptation of the anodic biofilm to the degradation of biowaste through metabolic interactions between microbial community members.
Keywords: Microbial fuel cell; Bioanode; Biowaste; Biofilm; Electroactive;

Influence of anode surface chemistry on microbial fuel cell operation by Carlo Santoro; Sofia Babanova; Kateryna Artyushkova; Jose A. Cornejo; Linnea Ista; Orianna Bretschger; Enrico Marsili; Plamen Atanassov; Andrew J. Schuler (141-149).
Self-assembled monolayers (SAMs) modified gold anodes are used in single chamber microbial fuel cells for organic removal and electricity generation. Hydrophilic (―N(CH3)3 +, ―OH, ―COOH) and hydrophobic (―CH3) SAMs are examined for their effect on bacterial attachment, current and power output. The different substratum chemistry affects the community composition of the electrochemically active biofilm formed and thus the current and power output. Of the four SAM-modified anodes tested, ―N(CH3)3 + results in the shortest start up time (15 days), highest current achieved (225 μA cm− 2) and highest MFC power density (40 μW cm− 2), followed by ―COOH (150 μA cm− 2 and 37 μW cm− 2) and ―OH (83 μA cm− 2 and 27 μW cm− 2) SAMs. Hydrophobic SAM decreases electrochemically active bacteria attachment and anode performance in comparison to hydrophilic SAMs (―CH3 modified anodes 7 μA cm− 2 anodic current and 1.2 μW cm− 2 MFC's power density). A consortium of Clostridia and δ-Proteobacteria is found on all the anode surfaces, suggesting a synergistic cooperation under anodic conditions.
Keywords: Microbial fuel cells; Self assembled monolayer; Surface modification; Bioelectrocatalysis; Anode biofilm analysis;

Geobacter-dominated biofilms can be selected under stringent conditions that limit the growth of competing bacteria. However, in many practical applications, such stringent conditions cannot be maintained and the efficacy and stability of these artificial biofilms may be challenged. In this work, biofilms were selected on low-potential anodes (− 0.36 V vs Ag/AgCl, i.e. − 0.08 V vs SHE) in minimal acetate or ethanol media. Selection conditions were then relaxed by transferring the biofilms to synthetic wastewater supplemented with soil as a source of competing bacteria. We tracked community succession and functional changes in these biofilms. The Geobacter-dominated biofilms showed stability in their community composition and electrochemical properties, with Geobacter sp. being still electrically active after six weeks in synthetic wastewater with power densities of 100 ± 19 mW·m− 2 (against 74 ± 14 mW·m− 2 at week 0) for all treatments. After six weeks, the ethanol-selected biofilms, despite their high taxon richness and their efficiency at removing the chemical oxygen demand (0.8 g·L− 1 removed against the initial 1.3 g·L− 1 injected), were the least stable in terms of community structure. These findings have important implications for environmental microbial fuel cells based on Geobacter-dominated biofilms and suggest that they could be stable in challenging environments.
Keywords: Geobacter biofilm; Microbial fuel cell; Wastewater; Functional stability; Bacterial community succession;

A comprehensive impedance journey to continuous microbial fuel cells by Surajbhan Sevda; Kudakwashe Chayambuka; T.R. Sreekrishnan; Deepak Pant; Xochitl Dominguez-Benetton (159-166).
The aim of the present work was to characterize the impedance response of an air-cathode MFC operating in a continuous mode and to determine intrinsic properties that define its performance which are crucial to be controlled for scalability purposes. The limiting step on electricity generation is the anodic electrochemically-active biofilm, independently of the external resistance, Rext, utilized. However, for Rext below 3 kΩ the internal impedance of the bioanode remained invariable, in good correspondence to the power density profile. The hydraulic retention time (HRT) had an effect on the impedance of both the bioanode and the air-cathode and especially on the overall MFC. The lowest HRT at which the MFC was operable was 3 h. Yet, the variation on the HRT did not have a significant impact on power generation. A two constant phase element-model was associated with the EIS response of both bioanode and air-cathode, respectively. Consistency was found between the CPE behaviour and the normal power-law distribution of local resistivity with a uniform dielectric constant, which represented consistent values with the electrical double layer, the Nernst diffusion layer and presumably the biofilm thickness. These results have future implications on MFC monitoring and control, as well as in providing critical parameters for scale-up.
Keywords: Air-cathode MFC; External resistance; Hydraulic retention time; Electrochemical impedance spectroscopy; Power density;

Monophyletic group of unclassified γ-Proteobacteria dominates in mixed culture biofilm of high-performing oxygen reducing biocathode by Michael Rothballer; Matthieu Picot; Tina Sieper; Jan B.A. Arends; Michael Schmid; Anton Hartmann; Nico Boon; Cees J.N. Buisman; Frédéric Barrière; David P.B.T.B. Strik (167-176).
Several mixed microbial communities have been reported to show robust bioelectrocatalysis of oxygen reduction over time at applicable operation conditions. However, clarification of electron transfer mechanism(s) and identification of essential micro-organisms have not been realised. Therefore, the objective of this study was to shape oxygen reducing biocathodes with different microbial communities by means of surface modification using the electrochemical reduction of two different diazonium salts in order to discuss the relation of microbial composition and performance. The resulting oxygen reducing mixed culture biocathodes had complex bacterial biofilms variable in size and shape as observed by confocal and electron microscopy. Sequence analysis of ribosomal 16S rDNA revealed a putative correlation between the abundance of certain microbiota and biocathode performance. The best performing biocathode developed on the unmodified graphite electrode and reached a high current density for oxygen reducing biocathodes at neutral pH (0.9 A/m2). This correlated with the highest domination (60.7%) of a monophyletic group of unclassified γ-Proteobacteria. These results corroborate earlier reports by other groups, however, higher current densities and higher presence of these unclassified bacteria were observed in this work. Therefore, members of this group are likely key-players for highly performing oxygen reducing biocathodes.
Keywords: Microbial fuel cell; Biofilm; Oxygen reduction; Biocathode; 454 amplicon sequencing;

This paper reviews the state-of-the art of the yeast-based biofuel cell research and development. The established extracellular electron transfer (EET) mechanisms in the presence and absence of exogenous mediators are summarized and discussed. The approaches applied for improvement of mediator-less yeast-based biofuel cells performance are also presented. The overview of the literature shows that biofuel cells utilizing yeasts as biocatalysts generate power density in the range of 20 to 2440 mW/m2, which values are comparable with the power achieved when bacteria are used instead. The electrons' origin and the contribution of the glycolysis, fermentation, aerobic respiration, and phosphorylation to the EET are commented. The reported enhanced current generation in aerobic conditions presumes reconsideration of some basic MFC principles. The challenges towards the practical application of the yeast-based biofuel cells are outlined.
Keywords: Yeast; Microbial fuel cells; Extracellular electron transfer; Mediators; Electricity generation;

Effects of atmospheric air plasma treatment of graphite and carbon felt electrodes on the anodic current from Shewanella attached cells by Monica Epifanio; Saikumar Inguva; Michael Kitching; Jean-Paul Mosnier; Enrico Marsili (186-193).
The attachment of electrochemically active microorganisms (EAM) on an electrode is determined by both the chemistry and topography of the electrode surface. Pre-treatment of the electrode surface by atmospheric air plasma introduces hydrophilic functional groups, thereby increasing cell attachment and electroactivity in short-term experiments. In this study, we use graphite and carbon felt electrodes to grow the model EAM Shewanella loihica PV-4 at oxidative potential (0.2 V vs. Ag/AgCl). Cell attachment and electroactivity are measured through electrodynamic methods. Atmospheric air plasma pre-treatment increases cell attachment and current output at graphite electrodes by 25%, while it improves the electroactivity of the carbon felt electrodes by 450%. Air plasma pre-treatment decreased the coulombic efficiency on both carbon felt and graphite electrodes by 60% and 80%, respectively. Microbially produced flavins adsorb preferentially at the graphite electrode, and air plasma pre-treatment results in lower flavin adsorption at both graphite and carbon felt electrodes. Results show that air plasma pre-treatment is a feasible option to increase current output in bioelectrochemical systems.
Keywords: Shewanella loihica PV-4; Atmospheric air plasma; Electroactivity;

A framework for modeling electroactive microbial biofilms performing direct electron transfer by Benjamin Korth; Luis F.M. Rosa; Falk Harnisch; Cristian Picioreanu (194-206).
A modeling platform for microbial electrodes based on electroactive microbial biofilms performing direct electron transfer (DET) is presented. Microbial catabolism and anabolism were coupled with intracellular and extracellular electron transfer, leading to biofilm growth and current generation. The model includes homogeneous electron transfer from cells to a conductive biofilm component, biofilm matrix conduction, and heterogeneous electron transfer to the electrode. Model results for Geobacter based anodes, both at constant electrode potential and in voltammetric (dynamic electrode potential) conditions, were compared to experimental data from different sources. The model can satisfactorily describe microscale (concentration, pH and redox gradients) and macroscale (electric currents, biofilm thickness) properties of Geobacter biofilms. The concentration of electrochemically accessible redox centers, here denominated as cytochromes, involved in the extracellular electron transfer, plays the key role and may differ between constant potential (300 mM) and dynamic potential (3 mM) conditions. Model results also indicate that the homogeneous and heterogeneous electron transfer rates have to be within the same order of magnitude (1.2 s− 1) for reversible extracellular electron transfer.Display Omitted
Keywords: Model; Bioelectrochemical systems; Electrochemically active microbial biofilms; Extracellular electron transfer; Microbial electrochemical technologies;

Long-term arsenic monitoring with an Enterobacter cloacae microbial fuel cell by Michelle Rasmussen; Shelley D. Minteer (207-212).
A microbial fuel cell was constructed with biofilms of Enterobacter cloacae grown on the anode. Bioelectrocatalysis was observed when the biofilm was grown in media containing sucrose as the carbon source and methylene blue as the mediator. The presence of arsenic caused a decrease in bioelectrocatalytic current. Biofilm growth in the presence of arsenic resulted in lower power outputs whereas addition of arsenic showed no immediate result in power output due to the short term arsenic resistance of the bacteria and slow transport of arsenic across cellular membranes to metabolic enzymes. Calibration curves plotted from the maximum current and maximum power of power curves after growth show that this system is able to quantify both arsenate and arsenate with low detection limits (46 μM for arsenate and 4.4 μM for arsenite). This system could be implemented as a method for long-term monitoring of arsenic concentration in environments where arsenic contamination could occur and alter the metabolism of the organisms resulting in a decrease in power output of the self-powered sensor.
Keywords: Bioelectrocatalysis; Microbial fuel cells; Arsenic detection; Enterobacter cloacae;

Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD+, respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP+, respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, − 90, and − 190 mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088 ± 0.022) and CNADPH/CNADP+ (0.268 ± 0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331 ± 0.094 for NAD and 1.96 ± 0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.
Keywords: Geobacter sulfurreducens; Adenosine phosphate; Nicotinamide adenine dinucleotide; Redox potential;

Specific and efficient electrochemical selection of Geoalkalibacter subterraneus and Desulfuromonas acetoxidans in high current-producing biofilms by Mélanie Pierra; Alessandro A. Carmona-Martínez; Eric Trably; Jean-Jacques Godon; Nicolas Bernet (221-225).
Two different saline sediments were used to inoculate potentiostatically controlled reactors (a type of microbial bioelectrochemical system, BES) operated in saline conditions (35 gNaCl  l− 1). Reactors were fed with acetate or a mixture of acetate and butyrate at two pH values: 7.0 or 5.5. Electroactive biofilm formation lag-phase, maximum current density production and coulombic efficiency were used to evaluate the overall performance of reactors. High current densities up to 8.5 A m− 2 were obtained using well-defined planar graphite electrodes. Additionally, biofilm microbial communities were characterized by CE-SSCP and 454 pyrosequencing. As a result of this procedure, two anode-respiring bacteria (ARB) always dominated the anodic biofilms: Geoalkalibacter subterraneus and/or Desulfuromonas acetoxidans. This suggests that a strong electrochemically driven selection process imposed by the applied potential occurs in the BES system. Moreover, the emergence of Glk. subterraneus in anodic biofilms significantly contributes to broaden the spectrum of high current producing microorganisms electrochemically isolated from environmental samples.Display Omitted
Keywords: Microbial bioelectrochemical systems; 454 pyrosequencing; Geoalkalibacter subterraneus; Desulfuromonas acetoxidans; Saline wastewater;

In this study, duckweed species Lemna valdiviana was investigated as a photoautotrophycally grown biocatalyst in recently developed Direct Photosynthetic Plant Fuel Cell. Stable current outputs, reaching maximum of 226 ± 11 mА/m2, were achieved during the operating period. The electricity production is associated with electrons generated through the light-dependent reactions in the chloroplasts as well as the respiratory processes in the mitochondria and transferred to the anode via endogenous electron shuttle, synthesized by the plants as a specific response to the polarization. In parallel, a considerable increase in the content of proteins (47%) and reserve carbohydrates (44%) of duckweeds grown under polarization conditions was established by means of biochemical analyses. This, combined with the electricity generation, makes the technology a feasible approach for the duckweed farming.Display Omitted
Keywords: Duckweeds; Lemna valdiviana; Direct Photosynthetic Plant Fuel Cell; Electricity generation; Protein and carbohydrates production;

The influence of mitochondrial electron transport chain inhibitors on the electricity outputs of Candida melibiosica yeast-based biofuel cell was investigated. The addition of 30 μM rotenone or antimycin A to the yeast suspension results in a decrease in the current generation, corresponding to 25.7 ± 1.3%, respectively 38.8 ± 1.9% reduction in the electric charge passed through the bioelectrochemical system. The latter percentage coincides with the share of aerobic respiration in the yeast catabolic processes, determined by the decrease of the ethanol production during cultivation in the presence of oxygen compared with that obtained under strict anaerobic conditions. It was established that the presence of both inhibitors leads to almost complete mitochondrial dysfunction, expressed by inactivation of cytochrome c oxidase and NADH:ubiquinone oxidoreductase as well as reduced electrochemical activity of isolated yeast mitochondria. It was also found that methylene blue partially neutralized the rotenone poisoning, probably serving as alternative intracellular electron shuttle for by-passing the complex I blockage. Based on the obtained results, we suppose that electrons generated through the aerobic respiration processes in the mitochondria participate in the extracellular electron transfer from the yeast cells to the biofuel cell anode, which contributes to higher current outputs at aerobic conditions.Display Omitted
Keywords: Yeast-based biofuel cell; Extracellular electron transfer; Mitochondria; Electron transport chains; Inhibition;

PTFE effect on the electrocatalysis of the oxygen reduction reaction in membraneless microbial fuel cells by Edoardo Guerrini; Matteo Grattieri; Alessio Faggianelli; Pierangela Cristiani; Stefano Trasatti (240-247).
Influence of PTFE in the external Gas Diffusion Layer (GDL) of open-air cathodes applied to membraneless microbial fuel cells (MFCs) is investigated in this work. Electrochemical measurements on cathodes with different PTFE contents (200%, 100%, 80% and 60%) were carried out to characterize cathodic oxygen reduction reaction, to study the reaction kinetics. It is demonstrated that ORR is not under diffusion-limiting conditions in the tested systems. Based on cyclic voltammetry, an increase of the cathodic electrochemical active area took place with the decrease of PTFE content. This was not directly related to MFC productivity, but to the cathode wettability and the biocathode development. Low electrodic interface resistances (from 1 to 1.5 Ω at the start, to near 0.1 Ω at day 61) indicated a negligible ohmic drop. A decrease of the Tafel slopes from 120 to 80 mV during productive periods of MFCs followed the biological activity in the whole MFC system. A high PTFE content in the cathode showed a detrimental effect on the MFC productivity, acting as an inhibitor of ORR electrocatalysis in the triple contact zone.The lowest PTFE content (60%) manifested mechanical instability of the cathode, together with the best performance.Display Omitted
Keywords: Microbial fuel cells; PTFE; Biocathode; Oxygen reduction; Gas Diffusion Layer;