Iron-Nicarbazin derived platinum group metal-free electrocatalyst in scalable-size air-breathing cathodes for microbial fuel cells.

In this work, a platinum group metal-free (PGM-free) catalyst based on iron as transitional metal and Nicarbazin (NCB) as low cost organic precursor was synthesized using Sacrificial Support Method (SSM). The catalyst was then incorporated into a large area air-breathing cathode fabricated by pressing with a large diameter pellet die. The electrochemical tests in abiotic conditions revealed that after a couple of weeks of successful operation, the electrode experienced drop in performances in reason of electrolyte leakage, which was not an issue with the smaller electrodes. A decrease in the hydrophobic properties over time and a consequent cathode flooding was suspected to be the cause. On the other side, in the present work, for the first time, it was demonstrated the proof of principle and provided initial guidance for manufacturing MFC electrodes with large geometric areas. The tests in MFCs showed a maximum power density of 1.85 W m-2. The MFCs performances due to the addition of Fe-NCB were much higher compared to the iron-free material. A numerical model using Nernst-Monod and Butler-Volmer equations were used to predict the effect of electrolyte solution conductivity and distance anode-cathode on the overall MFC power output. Considering the existing conditions, the higher overall power predicted was 3.6 mW at 22.2 S m-1 and at inter-electrode distance of 1 cm.

Modelling potential/current distribution in microbial electrochemical systems shows how the optimal bioanode architecture depends on electrolyte conductivity.

The theoretical bases for modelling the distribution of the electrostatic potential in microbial electrochemical systems are described. The secondary potential distribution (i.e. without mass transport limitation of the substrate) is shown to be sufficient to validly address microbial electrolysis cells (MECs). MECs are modelled with two different ionic conductivities of the solution (1 and 5.3 S m(-1)) and two bioanode kinetics (jmax = 5.8 or 34 A m(-2)). A conventional reactor configuration, with the anode and the cathode face to face, is compared with a configuration where the bioanode perpendicular to the cathode implements the electrochemical reaction on its two sides. The low solution conductivity is shown to have a crucial impact, which cancels out the advantages obtained by setting the bioanode perpendicular to the cathode. For the same reason, when the surface area of the anode is increased by multiplying the number of plates, care must be taken not to create too dense anode architecture. Actually, the advantages of increasing the surface area by multiplying the number of plates can be lost through worsening of the electrochemical conditions in the multi-layered anode, because of the increase of the electrostatic potential of the solution inside the anode structure. The model gives the first theoretical bases for scaling up MECs in a rather simple but rigorous way.

Electrochemical assay of mammalian cell viability.

We present the first use of amperometric detection to assess the viability of mammalian cells in continuous mode, directly in the cell culture medium. Vero or HeLa cells were injected into electrochemical sensors equipped with a 3-electrode system and containing DCIP 50 µM used as the redox mediator. DCIP was reduced by the viable cells and the reduced form was detected amperometrically at 300 mV vs silver pseudo-reference. The continuous regeneration of the oxidized form of the mediator ensured a stable redox state of the cell environment, allowing the cells to survive during the measurement time. The electrochemical response was related to cell metabolism (no response with dead cells or lysed cells) and depended on both mediator concentration and cell density. The protocol was applied to both cells in suspension and adhered cells. It was also adapted to detect trans-plasma membrane electron transfer (tPMET) by replacing DCIP by ferricyanide 500 µM and using linear scan voltammetry (2 mV/s). The pioneering results described here pave the way to the development of routine electrochemical assays for cell viability and for designing a cell-based analytical platform.

Direct electrochemical detection of trans-plasma membrane electron transfer: A possible alternative pathway for cell respiration.

An electrochemical protocol was designed to enable Vero cells to transfer electrons to an electrode without any added redox mediator. The cells were cultured on the surface of electrodes polarized at the optimal potential of 400 mV/silver pseudo-reference. Gold, carbon, and CNT-coated carbon electrodes displayed similar current record patterns. Extracellular electron transfer was sustained for several days. Its intensity, up to 1.5 pA.cell-1, was in the range of the electron flows implemented by cell respiration. A large fraction of the current vanished as soon as anoxic conditions were established, which suggests a mitochondrial origin for a large proportion of the electrons. The current records always showed a two-phase pattern. The occurrence of the two phases was not due to an evolution of the cell mat structure, which was fully established during the first day of polarization and did not change significantly thereafter. Increasing the cell seeding density decreased the maximum current reached during the first phase and the duration of the phase. These observations put together lead us to propose a model, in which only the cells adhered on the electrode surface produced current by metabolizing glutamine during the first phase. The possible role of this extracellular electron transfer as an alternative cell respiration pathway is discussed. The key roles it could play in regulating pH and pO2 gradients are considered, specifically to explain the pH gradient reversal observed in cancer cells. These pioneering results pave the way for electrochemical sensors to directly address cellular metabolic pathways.

Towards an engineering-oriented strategy for building microbial anodes for microbial fuel cells.

The objective of the work was to give some first insight into an engineering-oriented approach to MFC design by focusing on anode optimisation. The effect of various parameters was firstly investigated in half cell set-ups under well-controlled conditions. Microbial anodes were formed from soil leachate under polarisation at -0.2 V vs. SCE with different concentrations of substrate, salt and buffer. It was shown that non-turnover CV could be used to assess the electroactive maturity of the anodes during polarisation. This first phase resulted in the definition of a set of optimal parameter values. In the second phase, an optimal anode was formed in a half-cell under the defined optimal conditions. A numerical approach was then developed to calculate the theoretical maximum power that the anode could provide in an ideal MFC. The concept of "ideal MFC" introduced here allowed the theoretical maximum power to be calculated on the sole basis of the kinetic characteristics of the anode. Finally, a MFC designed in the aim of approaching such ideal conditions generated stable power densities of 6.0 W m(-2), which were among the highest values reported so far. The discrepancy between the theoretical maximum (8.9 W m(-2)) and the experimental results pointed out some limit due to the source of inoculum and suggested possible paths to improvement.

The electrochemical potential is a key parameter for cell adhesion and proliferation on carbon surface.

The Nernst potential of the support/cell interface is suspected to play a key role in cell adhesion and proliferation. However, the studies that have addressed this topic have generally varied the electrochemical potential of the interface by comparing different materials or by varying the chemical composition of the surface coating. It is consequently hard to definitively separate the actual effect of the potential from possible side-effects due to differences in the surface composition or topography. Here, a 3-electrode set-up was used to apply different values of potential to identical carbon electrodes. Potentials were applied in the range -200 to 400 mV vs. silver pseudo-reference (SPR), i.e. 90 to 690 mV/SHE, to screen-printed carbon electrodes used to grow Vero or Raw 264.7 cell lines. Values up to 200 mV/SPR prohibited cell adhesion and even caused detachment of cells that were previously adhered. The value of 400 mV/DRP allowed cell adhesion and proliferation, leading to confluent and sometimes very compact mats. The zero charge potential, measured around 200 mV/DRP, showed that the drastic effect of the applied potential was probably due to the negative/positive switch of the surface charge.

Electroactivity of phototrophic river biofilms and constitutive cultivable bacteria.

Electroactivity is a property of microorganisms assembled in biofilms that has been highlighted in a variety of environments. This characteristic was assessed for phototrophic river biofilms at the community scale and at the bacterial population scale. At the community scale, electroactivity was evaluated on stainless steel and copper alloy coupons used both as biofilm colonization supports and as working electrodes. At the population scale, the ability of environmental bacterial strains to catalyze oxygen reduction was assessed by cyclic voltammetry. Our data demonstrate that phototrophic river biofilm development on the electrodes, measured by dry mass and chlorophyll a content, resulted in significant increases of the recorded potentials, with potentials of up to +120 mV/saturated calomel electrode (SCE) on stainless steel electrodes and +60 mV/SCE on copper electrodes. Thirty-two bacterial strains isolated from natural phototrophic river biofilms were tested by cyclic voltammetry. Twenty-five were able to catalyze oxygen reduction, with shifts of potential ranging from 0.06 to 0.23 V, cathodic peak potentials ranging from -0.36 to -0.76 V/SCE, and peak amplitudes ranging from -9.5 to -19.4 µA. These isolates were diversified phylogenetically (Actinobacteria, Firmicutes, Bacteroidetes, and Alpha-, Beta-, and Gammaproteobacteria) and exhibited various phenotypic properties (Gram stain, oxidase, and catalase characteristics). These data suggest that phototrophic river biofilm communities and/or most of their constitutive bacterial populations present the ability to promote electronic exchange with a metallic electrode, supporting the following possibilities: (i) development of electrochemistry-based sensors allowing in situ phototrophic river biofilm detection and (ii) production of microbial fuel cell inocula under oligotrophic conditions.

From microbial fuel cell (MFC) to microbial electrochemical snorkel (MES): maximizing chemical oxygen demand (COD) removal from wastewater.

The paper introduces the concept of the microbial electrochemical snorkel (MES), a simplified design of a "short-circuited" microbial fuel cell (MFC). The MES cannot provide current but it is optimized for wastewater treatment. An electrochemically active biofilm (EAB) was grown on graphite felt under constant polarization in an urban wastewater. Controlling the electrode potential and inoculating the bioreactor with a suspension of an established EAB improved the performance and the reproducibility of the anodes. Anodes, colonized by an EAB were tested for the chemical oxygen demand (COD) removal from urban wastewater using a variety of bio-electrochemical processes (microbial electrolysis, MFC, MES). The MES technology, as well as a short-circuited MFC, led to a COD removal 57% higher than a 1000 Ω-connected MFC, confirming the potential for wastewater treatment.

Theoretical analysis of the electrochemical systems used for the application of direct current/voltage stimuli on cell cultures.

Endogenous electric fields drive many essential functions relating to cell proliferation, motion, differentiation and tissue development. They are usually mimicked in vitro by using electrochemical systems to apply direct current or voltage stimuli to cell cultures. The many studies devoted to this topic have given rise to a wide variety of experimental systems, whose results are often difficult to compare. Here, these systems are analysed from an electrochemical standpoint to help harmonize protocols and facilitate optimal understanding of the data produced. The theoretical analysis of single-electrode systems shows the necessity of measuring the Nernst potential of the electrode and of discussing the results on this basis rather than using the value of the potential gradient. The paper then emphasizes the great complexity that can arise when high cell voltage is applied to a single electrode, because of the possible occurrence of anode and cathode sites. An analysis of two-electrode systems leads to the advice to change experimental practices by applying current instead of voltage. It also suggests that the values of electric fields reported so far may have been considerably overestimated in macro-sized devices. It would consequently be wise to revisit this area by testing considerably lower electric field values.

Catalysis of the electrochemical oxygen reduction reaction (ORR) by animal and human cells.

Animal cells from the Vero lineage and MRC5 human cells were checked for their capacity to catalyse the electrochemical oxygen reduction reaction (ORR). The Vero cells needed 72 hours' incubation to induce ORR catalysis. The cyclic voltammetry curves were clearly modified by the presence of the cells with a shift of ORR of 50 mV towards positive potentials and the appearance of a limiting current (59 µA.cm-2). The MRC5 cells induced considerable ORR catalysis after only 4 h of incubation with a potential shift of 110 mV but with large experimental deviation. A longer incubation time, of 24 h, made the results more reproducible with a potential shift of 90 mV. The presence of carbon nanotubes on the electrode surface or pre-treatment with foetal bovine serum or poly-D-lysine did not change the results. These data are the first demonstrations of the capability of animal and human cells to catalyse electrochemical ORR. The discussion of the possible mechanisms suggests that these pioneering observations could pave the way for electrochemical biosensors able to characterize the protective system of cells against oxidative stress and its sensitivity to external agents.

How bacteria use electric fields to reach surfaces.

Electrotaxis is the property of cells to sense electric fields and use them to orient their displacement. This property has been widely investigated with eukaryotic cells but it remains unclear whether or not bacterial cells can sense an electric field. Here, a specific experimental set-up was designed to form microbial electroactive biofilms while differentiating the effect of the electric field from that of the polarised electrode surface. Application of an electric field during exposure of the electrodes to the inoculum was shown to be required for an electroactive biofilm to form afterwards. Similar biofilms were formed in both directions of the electric field. This result is attributed to the capacity of the cells to detect the K+ and Na+ ion gradients that the electric field creates at the electrode surface. This microbial property should now be considered as a key factor in the formation of electroactive biofilms and possible implications in the biomedical domain are discussed.

Oxygen-reducing microbial cathodes in hypersaline electrolyte.

Hypersaline electrolytes offer a way to boost the development of microbial fuel cells by overcoming the issue due to the low conductivity of the usual media. Efficient halotolerant bioanodes have already been designed but O2-reducing cathodes remain a strong bottleneck. Here, O2-reducing biocathodes were designed by using salt marsh sediment as the inoculum and a hypersaline media (45 g/L NaCl) of high conductivity (10.4 S m-1). Current density up to 2.2 A m-2 was reached from potential of +0.2 V/SCE. The efficiency of the biocathodes was correlated to the presence of Gammaproteobacteria strain(s) related to Thiohalobacter thiocyanaticus, which were considerably enriched in the best performing biocathodes. This work opens up new perspectives to overcome the O2 reduction issue in hypersaline MFCs by designing efficient halotolerant microbial cathodes and pointing out the strains that should now be focused to improve them.

Oxygen-reducing bidirectional microbial electrodes designed in real domestic wastewater.

Microbial electrodes were designed in domestic wastewaters to catalyse the oxidation of organic matter (anode) and the reduction of oxygen (cathode) alternately. The successive aeration phases (cathode) enhanced the anodic efficiency, resulting in current densities of up to 6.4 Am-2 without the addition of any substrate. Using nitrogen during the anodic phases affected the microbial populations and the electrodes showed a lower ability to subsequently turn to O2 reduction than the microbial anodes formed in open-to-air conditions did. No strong difference was observed between internal and external biofilm, both of which showed a very large variety of taxa in terms of abundance as well as variance. They comprised a mix of aerobic and anaerobic species, many of which have already been identified separately in bioelectrochemical systems. Such a large diversity, which had not been observed in aerobic bidirectional bioelectrodes so far, can explain the efficiency and robustness observed here.

Hypersaline microbial fuel cell equipped with an oxygen-reducing microbial cathode.

Microbial anodes and oxygen reducing microbial cathodes were designed separately under constant polarization at + 0.1 V/SCE in a hypersaline medium (NaCl 45 g/L). They were then associated to design two-compartment microbial fuel cells (MFCs). These MFCs produced up to 209 ± 24 mW m-2 during a week. This was the first demonstration that hypersaline MFCs equipped with microbial cathodes can produce power density at this level. Desulfuromonas sp. were confirmed to be key species of the anodes. The efficiency of the cathodes was linked to the development of a redox system centred at + 0.2 V/SCE and to the presence of Gammaproteobacteria (Alteromonadales and Oceanospirillales), especially an unclassified order phylogenetically linked to the genus Thioalobacter. Comparing the different performance of the four MFCs with the population analyses suggested that polarization at + 0.1 V/SCE should be maintained longer to promote the growth of Thioalobacter on the cathode and thus increase the MFC performance.

Industrially scalable surface treatments to enhance the current density output from graphite bioanodes fueled by real domestic wastewater.

Acid and electrochemical surface treatments of graphite electrode, used individually or in combination, significantly improved the microbial anode current production, by +17% to +56%, in well-regulated and duplicated electroanalytical experimental systems. Of all the consequences induced by surface treatments, the modifications of the surface nano-topography preferentially justify an improvement in the fixation of bacteria, and an increase of the specific surface area and the electrochemically accessible surface of graphite electrodes, which are at the origin of the higher performances of the bioanodes supplied with domestic wastewater. The evolution of the chemical composition and the appearance of C-O, C=O, and O=C-O groups on the graphite surface created by combining acid and electrochemical treatments was prejudicial to the formation of efficient domestic-wastewater-oxidizing bioanodes. The comparative discussion, focused on the positioning of the performances, shows the industrial interest of applying the surface treatment method to the world of bioelectrochemical systems.

How Comparable are Microbial Electrochemical Systems around the Globe? An Electrochemical and Microbiological Cross-Laboratory Study.

A cross-laboratory study on microbial fuel cells (MFC) which involved different institutions around the world is presented. The study aims to assess the development of autochthone microbial pools enriched from domestic wastewater, cultivated in identical single-chamber MFCs, operated in the same way, thereby approaching the idea of developing common standards for MFCs. The MFCs are inoculated with domestic wastewater in different geographic locations. The acclimation stage and, consequently, the startup time are longer or shorter depending on the inoculum, but all MFCs reach similar maximum power outputs (55±22 µW cm-2 ) and COD removal efficiencies (87±9 %), despite the diversity of the bacterial communities. It is inferred that the MFC performance starts when the syntrophic interaction of fermentative and electrogenic bacteria stabilizes under anaerobic conditions at the anode. The generated power is mostly limited by electrolytic conductivity, electrode overpotentials, and an unbalanced external resistance. The enriched microbial consortia, although composed of different bacterial groups, share similar functions both on the anode and the cathode of the different MFCs, resulting in similar electrochemical output.

How Comparable are Microbial Electrochemical Systems around the Globe? An Electrochemical and Microbiological Cross-Laboratory Study.

Invited for this month's cover is the collaborative work among Univ. of Milano-Bicocca, Ricerca sul Sistema Energetico S.p.A., Univ. degli Studi di Milano, Univ. of California Irvine, Univ. of New Mexico, CNRS Toulouse. Technische Univ. Braunschweig, Aquacycl LLC, J. Craig Venter Institute, Helmholtz-Centre for Environmental Research. The image shows a sketch of a microbial fuel cell and a target indicating the need of developing common standards for the field of microbial electrochemical technologies. The Full Paper itself is available at 10.1002/cssc.202100294.

Effect of pore size on the current produced by 3-dimensional porous microbial anodes: A critical review.

Microbial anodes are the cornerstone of most electro-microbial processes. Designing 3-dimensional porous electrodes to increase the surface area of the electroactive biofilm they support is a key challenge in order to boost their performance. In this context, the critical review presented here aims to assess whether an optimal range of pore size may exist for the design of microbial anodes. Pore sizes of a few micrometres can enable microbial cells to penetrate but in conditions that do not favour efficient development of electroactive biofilms. Pores of a few tens of micrometres are subject to clogging. Sizes of a few hundreds of micrometres allow penetration of the biofilm inside the structure, but its development is limited by internal acidification. Consequently, pore sizes of a millimetre or so appear to be the most suitable. In addition, a simple theoretical approach is described to establish basis for porous microbial anode design.

Effect of surface roughness, porosity and roughened micro-pillar structures on the early formation of microbial anodes.

The early formation of electroactive biofilms was investigated with gold electrodes inoculated with Geobacter sulfurreducens. Biofilms were formed under an applied potential of 0.1 V/SCE, with a single batch of acetate 10 mM, on flat gold electrodes with different random surface roughness. Roughness with arithmetical mean height (Sa) ranging from 0.5 to 6.7 µm decreased the initial latency time, and increased the current density by a factor of 2.7 to 6.7 with respect to nano-rough electrodes (Sa = 4.5 nm). The current density increased linearly with Sa up to 14.0 A·m-2 for Sa of 6.7 µm. This linear relationship remained valid for porous gold. In this case, the biofilm rapidly formed a uniform layer over the pores, so porosity impacted the current only by modifying the roughness of the upper surface. The current density thus reached 14.8 ±â€¯1.1 A·m-2 with Sa of 7.6 µm (7 times higher than the nano-rough electrodes). Arrays of 500-µm-high micro-pillars were roughened following the same protocol. In this case, roughening resulted in a modest gain around 1.3-fold. A numerical model showed that the modest enhancement was due to ion transport not being sufficient to mitigate the local acidification of the structure bottom.

Forming electrochemically active biofilms from garden compost under chronoamperometry.

Dimensionally stable anodes (DSA) were polarized at different constant potential values for several days in garden compost. After an initial lag period ranging from 1 to 10.5 days, the current increased fast and then stabilized for days. Current densities higher than 100 mA m(-2) and up to 385 mA m(-2) were obtained with the sole organic matter contained in compost as substrate. Control experiments performed with sterilized compost, oscillations of the current with the temperature, kinetics of the exponential phase of current increase and observations of the surface of electrodes by epifluorescence microscopy showed that the current was controlled by the colonization of the electrode surface by a biofilm which originated the indigenous flora of compost. Three individually addressed electrodes polarized at different potentials in the same reactor led to identical current evolutions on each electrode, which underlined the key role of the microbial flora of the compost in the discrepancy observed in the other experiments. Chronoamperometry revealed a promising technique to check natural environments for new electrochemically active microbial species.