Keith Baronian

Bacteria and yeasts as catalysts in microbial fuel cells: electron transfer from micro-organisms to electrodes for green electricity

This article reviews the use of micro-organisms as catalysts at the electrodes of microbial fuel cells (MFCs). The principle of MFCs and their intended use for water treatment and clean electricity production is discussed. We address the different microbial structure and metabolic pathways found in prokaryote (bacteria) and eukaryote (yeasts) that allow the understanding of why electron transfer is possible between a microbe and an electrode. The different mechanisms of microbe–electrode electron transfer are discussed: direct electron transfer or through natural nanowires (pili), mediated electron transfer by natural or artificial redox mediator and finally direct redox transformation of excreted metabolites at the electrodes. This is followed by a review of the different bacteria that have been found and studied in MFCs mainly in the anodic compartment but also more recently in the cathodic side of the fuel cells. A perspective on the possible advantages and challenges of the use of yeasts in MFCs is provided, as this aspect has not been thoroughly studied so far. The fourth section of the review focuses on how to improve the performance and sustainability of MFCs through the functionalisation of the electrode surface, for instance with the covalent grafting of redox mediators and/or enzymes.

Biochemical mediator demand--a novel rapid alternative for measuring biochemical oxygen demand.

Abstract The biochemical oxygen demand (BOD) test (BOD5) is a crucial environmental index for monitoring organic pollutants in waste water but is limited by the 5-day requirement for completing the test. We have optimised a rapid microbial technique for measuring the BOD of a standard BOD5 substrate (150 mg glucose/l, 150 mg glutamic acid/l) by quantifying an equivalent biochemical mediator demand in the absence of oxygen. Elevated concentrations of Escherichia coli were incubated with an excess of redox mediator, potassium hexacyanoferrate(III), and a known substrate for 1 h at 37 °C without oxygen. The addition of substrate increased the respiratory activity of the microorganisms and the accumulation of reduced mediator; the mediator was subsequently re-oxidised at a working electrode generating a current quantifiable by a coulometric transducer. Catabolic conversion efficiencies exceeding 75% were observed for the oxidation of the standard substrate. The inclusion of a mediator allowed a higher co-substrate concentration compared to oxygen and substantially reduced the incubation time from 5 days to 1 h. The technique replicates the traditional BOD5 method, except that a mediator is substituted for oxygen, and we aim to apply the principle to measure the BOD of real waste streams in future work.

Detection of two distinct substrate-dependent catabolic responses in yeast cells using a mediated electrochemical method

Abstract. Mediated electrochemical detection of catabolism in prokaryotic cells is well documented; however, the application of this technique to eukaryotic cells has received less attention. Two catabolic substrate-dependent mediated electrochemical signals were detected in the yeast Saccharomyces cerevisiae. The signal using a single hydrophilic mediator (ferricyanide) is small whereas the response using a double mediator system comprising a hydrophilic and a lipophilic mediator (ferricyanide and menadione) is up to three orders of magnitude larger. The behaviour of each response during cell ageing is different: the single mediator response increases whereas the double mediator response decreases. This difference indicates that the two signals originate at different points in the catabolic pathways. In S. cerevisiae the double mediator response is proposed to originate from the reduction of the lipophilic mediator by NADPH produced in the pentose phosphate pathway. The single mediator signal arises from reduction of the hydrophilic mediator by an extracellular redox species produced in response to the presence of glucose.

Characterisation of yeast microbial fuel cell with the yeast Arxula adeninivorans as the biocatalyst.

Yeast microbial fuel cells have received little attention to date. Yeast should be ideal MFC catalyst because they are robust, easily handled, mostly non-pathogenic organisms with high catabolic rates and in some cases a broad substrate spectrum. Here we show that the non-conventional yeast Arxula adeninvorans transfers electrons to an electrode through the secretion of a reduced molecule that is not detectable when washed cells are first resuspended but which accumulates rapidly in the extracellular environment. It is a single molecule that accumulates to a significant concentration. The occurrence of mediatorless electron transfer was first established in a conventional microbial fuel cell and that phenomenon was further investigated by a number of techniques. Cyclic voltammetry (CV) on a yeast pellet shows a single peak at 450 mV, a scan rate study showed that the peak was due to a solution species. CVs of the supernatant confirmed a solution species. It appears that, given its other attributes, A. adeninivorans is a good candidate for further investigation as a MFC catalyst.

Electrochemical detection of intracellular and cell membrane redox systems in Saccharomyces cerevisiae

Redox mediators can interact with eukaryote cells at a number of different cell locations. While cell membrane redox centres are easily accessible, the redox centres of catabolism are situated within the cytoplasm and mitochondria and can be difficult to access. We have systematically investigated the interaction of thirteen commonly used lipophilic and hydrophilic mediators with the yeast Saccharomyces cerevisiae. A double mediator system is used in which ferricyanide is the final electron acceptor (the reporter mediator). After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells. The plateau current at 425 mV vs Ag/AgCl gives the analytical signal. The results show that five of the mediators interact with at least three different trans Plasma Membrane Electron Transport systems (tPMETs), and that four mediators cross the plasma membrane to interact with cytoplasmic and mitochondrial redox molecules. Four of the mediators inhibit electron transfer from S. cerevisiae. Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

Evaluation of a thermophile enzyme for a carbon paste amperometric biosensor: L-glutamate dehydrogenase

Abstract An amperometric L-glutamate sensor was prepared by the immobilization of thermophilic L-glutamate dehydrogenase (L-glutamate: NADP + oxido-reductase, deaminating and transaminating, EC 1.4.1.4.) in a carbon paste working electrode with a polyethylenimine Toluidine Blue O redox polymer mediator and lactitol/DEAE dextran stabilizing system. The response of the sensor was both pH and temperature dependent. Decreases in K m app (mM) and increases in V max (μA) were observed with increasing temperature, whilst increasing pH resulted in increases in both K m app and V max parameters. The measurements were based on the amperometric detection of the product of the enzymatic reaction, NADPH, via mediated reoxidation to NADP + at E app = 100 mV versus Ag/AgCl. The major characteristics of this method are its simplicity and reproducibility, although at higher temperatures (333 K and above) the properties of the carbon paste matrix itself limited the physical stability of the system.

Bioelectrochemical probing of intracellular redox processes in living yeast cells-application of redox polymer wiring in a microfluidic environment

AbstractConventionally, microbial bioelectrochemical assays have been conducted using immobilized cells on an electrode that is placed in an electrochemical batch cell. In this paper, we describe a developed microfluidic platform with integrated microelectrode arrays for automated bioelectrochemical assays utilizing a new double mediator system to map redox metabolism and screen for genetic modifications in Saccharomyces cerevisiae cells. The function of this new double mediator system based on menadione and osmium redox polymer (PVI-Os) is demonstrated. “Wiring” of S. cerevisiae cells using PVI-Os shows a significant improvement of bioelectrochemical monitoring in a microfluidic environment and functions as an effective immobilization matrix for cells that are not strongly adherent. The function of the developed microfluidic platform is demonstrated using two strains of S. cerevisiae, ENY.WA and its deletion mutant EBY44, which lacks the enzyme phosphoglucose isomerase. The cellular responses to introduced glucose and fructose were recorded for the two S. cerevisiae strains, and the obtained results are compared with previously published work when using an electrochemical batch cell, indicating that microfluidic bioelectrochemical assays employing the menadione–PVI-Os double mediator system provides an effective means to conduct automated microbial assays. FigureMicrofluidic platform for bioelectrochemical assays using osmium redox polymer “wired” living yeast cells

Estimation of the Glomus intraradices nuclear DNA content

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Electron transfer from Proteus vulgaris to a covalently assembled, single walled carbon nanotube electrode functionalised with osmium bipyridine complex: application to a whole cell biosensor.

We report the fabrication and use of electrodes constructed from single walled carbon nanotubes (SWCNTs) chemically assembled on a carbon surface and functionalised with an osmium(II) bipyridine complex (Osbpy). The ability of the electrodes to transduce biologically generated currents from Proteus vulgaris has been established. Our investigations show that there are two contributions to the current: one from electroactive species secreted into solution and another from cell redox sites. The modified electrode can be used to monitor cell metabolism, thereby acting as a whole cell biosensor. The biosensor was used in a 1-h assay to investigate the toxicity of ethanol, sodium azide and the antibiotic ampicillin and gave quantitative data that were closely correlated with standard cell plate viability assays. The results provide proof of principle that the whole cell biosensor could be used for high throughput screening of antimicrobial activity. One of the modified electrodes was used for approximately 1000 measurements over four months demonstrating the robustness of the system.

Patterned arrays of vertically aligned carbon nanotube microelectrodes on carbon films prepared by thermal chemical vapor deposition.

A straightforward procedure is described for preparation of arrays of microdisk electrodes comprising bundles of vertically aligned carbon nanotubes (VACNTs). The arrays are fabricated by thermal chemical vapor deposition synthesis directly on a planar carbon film support. Use of standard micro- and nanolithography procedures for patterning the bilayer catalyst spots enables arrays to be grown with controlled electrode diameters and spacings. The minimum accessible VACNT bundle diameter, and hence microelectrode diameter, is 2 microm. After insulating the arrays with SU-8 epoxy and exposing the VACNT ends by polishing or treating with O2 plasma, the microdisk electrodes exhibit attractive electrochemical properties.