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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

Impact of electrode micro- and nano-scale topography on the formation and performance of microbial electrodes.

From a fundamental standpoint, microbial electrochemistry is unravelling a thrilling link between life and materials. Technically, it may be the source of a large number of new processes such as microbial fuel cells for powering remote sensors, autonomous sensors, microbial electrolysers and equipment for effluent treatment. Microbial electron transfers are also involved in many natural processes such as biocorrosion. In these contexts, a huge number of studies have dealt with the impact of electrode materials, coatings and surface functionalizations but very few have focused on the effect of the surface topography, although it has often been pointed out as a key parameter impacting the performance of electroactive biofilms. The first part of the review gives an overview of the influence of electrode topography on abiotic electrochemical reactions. The second part recalls some basics of the effect of surface topography on bacterial adhesion and biofilm formation, in a broad domain reaching beyond the context of electroactivity. On these well-established bases, the effect of surface topography is reviewed and analysed in the field of electroactive biofilms. General trends are extracted and fundamental questions are pointed out, which should be addressed to boost future research endeavours. The objective is to provide basic guidelines useful to the widest possible range of research communities so that they can exploit surface topography as a powerful lever to improve, or to mitigate in the case of biocorrosion for instance, the performance of electrode/biofilm interfaces.

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.

Effect of surface nano/micro-structuring on the early formation of microbial anodes with Geobacter sulfurreducens: Experimental and theoretical approaches.

Smooth and nano-rough flat gold electrodes were manufactured with controlled Ra of 0.8 and 4.5nm, respectively. Further nano-rough surfaces (Ra 4.5nm) were patterned with arrays of micro-pillars 500µm high. All these electrodes were implemented in pure cultures of Geobacter sulfurreducens, under a constant potential of 0.1V/SCE and with a single addition of acetate 10mM to check the early formation of microbial anodes. The flat smooth electrodes produced an average current density of 0.9A·m-2. The flat nano-rough electrodes reached 2.5A·m-2 on average, but with a large experimental deviation of ±2.0A·m-2. This large deviation was due to the erratic colonization of the surface but, when settled on the surface, the cells displayed current density that was directly correlated to the biofilm coverage ratio. The micro-pillars considerably improved the experimental reproducibility by offering the cells a quieter environment, facilitating biofilm development. Current densities of up to 8.5A·m-2 (per projected surface area) were thus reached, in spite of rate limitation due to the mass transport of the buffering species, as demonstrated by numerical modelling. Nano-roughness combined with micro-structuring increased current density by a factor close to 10 with respect to the smooth flat surface.

Different methods used to form oxygen reducing biocathodes lead to different biomass quantities, bacterial communities, and electrochemical kinetics.

Six biocathodes catalyzing oxygen reduction were designed from the same environmental inoculum but using three different methods. Two were formed freely at open circuit potential, two using conventional aerobic polarization at -0.2V/SCE and two by reversion of already established acetate-fed bioanodes. Observation of the biofilms by SEM and epifluorescence microscopy revealed that reversible bioelectrodes had the densest biofilms. Electrochemical characterization revealed two different redox systems for oxygen reduction, at -0.30 and +0.23V/SCE. The biocathodes formed under aerobic polarization gave higher electrocalatytic performance for O2 reduction, due to production of the redox systems at +0.23V/SCE. Analyses of the bacterial communities on the biocathodes by 16S-rRNA pyrosequencing showed different selection (or enrichment) of microorganisms depending on the method used. This study highlights how the method chosen for designing oxygen biocathodes can affect the cathode coverage, the selection of bacterial populations and the electrochemical performance.

Removable air-cathode to overcome cathode biofouling in microbial fuel cells.

An innovative microbial fuel cell (MFC) design is described, which allows the air-cathode to be replaced easily without draining the electrolyte. MFCs equipped with 9-cm2 or 50-cm2 bioanodes provided 0.6 and 0.7W/m2 (referred to the cathode surface area) and were boosted to 1.25 and 1.96W/m2, respectively, when the initial air-cathode was replaced by a new one. These results validate the practical interest of removable air-cathodes and evidence the importance of the cathode biofouling that takes place during the MFC starting phase. As this biofouling is compensated by the concomitant improvement of the bioanodes it cannot be detected on the power curves and may be a widespread cause of performance underestimation.

Halotolerant bioanodes: The applied potential modulates the electrochemical characteristics, the biofilm structure and the ratio of the two dominant genera.

The development of economically-efficient microbial electrochemical technologies remains hindered by the low ionic conductivity of the culture media used as the electrolyte. To overcome this drawback, halotolerant bioanodes were designed with salt marsh sediment used as the inoculum in electrolytes containing NaCl at 30 or 45g/L (ionic conductivity 7.0 or 10.4S·m(-1)). The bioanodes were formed at four different potentials -0.4, -0.2, 0.0 and 0.2V/SCE to identify the effect on the electrochemical kinetic parameters, the biofilm structures and the composition of the microbial communities. The bioanodes formed at -0.4V/SCE were largely dominated by Marinobacter spp. Voltammetry showed that they provided higher currents than the other bioanodes in the range of low potentials, but the maximum currents were limited by the poor surface colonization. The bioanodes formed at -0.2, 0.0 and 0.2V/SCE showed similar ratios of Marinobacter and Desulfuromonas spp. and higher values of the maximum current density. The combined analysis of kinetic parameters, biofilm structure and biofilm composition showed that Marinobacter spp., which ensured a higher electron transfer rate, were promising species for the design of halotolerant bioanodes. The challenge is now to overcome its limited surface colonization in the absence of Desulfuromonas spp.