Direct electron transfer with yeast cells and construction of a mediatorless microbial fuel cell

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Abstract

The direct electron transfer exhibited by the yeast cells, Hansenula anomala has been demonstrated using the electrochemical technique cyclic voltammetry by immobilizing the microorganisms by two different methods viz., physical adsorption and covalent linkage. The analysis of redox enzymes present in the outer membrane of the microorganisms has been carried out in this work. This paper demonstrates that yeast cells with redox enzymes present in their outer membrane are capable of communicating directly with the electrode surface and contribute to current generation in a mediatorless biofuel cells. The efficiency of current generation has been evaluated using three anode materials.

Introduction

Microbial fuel cells (MFCs) are bioelectrochemical transducers that convert microbial reducing power (generated by the metabolism of organic substrates) into electrical energy. The recent interest in microbial fuel cells is attributed to the possibility of combining waste degradation with energy generation (Shukla et al., 2004, Rabaey and Vestraete, 2005, Heydorn and Gee, 2004). Microorganisms can transfer electrons to the anode electrode in three ways; viz., (i) using exogenous mediators such as potassium ferricyanide, thionine or neutral red; (ii) using mediators produced by the bacteria themselves; (iii) direct transfer of electrons from inside of the bacterial cells (Leropoulos et al., 2005). There are several drawbacks of using exogenous mediators such as their high cost, short lifetime and toxicity to the microorganisms. However when the bacteria produce their own mediators or they transfer electrons directly to the electrode, the system can operate at a high-sustained level of activity.

Organisms such as Shewanella putrefaciens, Geobacter sulfuurreducens, Geobacter metalli reducens and Rhodoferax ferrireducens have been shown to generate electricity in mediatorless MFC systems. Some of the species that belong to the genera Geobacter, Geovibrio, Shewanella, reduce Fe(III) through their respiratory, fermentative or photosynthetic metabolism (Holmes et al., 2004a). Some of them are able to conserve the energy for growth by coupling the oxidation of organic acids, aromatic hydrocarbons and H2 to Fe(III) reduction or Mn(IV) reduction (Lovely and Philips, 1988, Lee et al., 1999). The Fe(III) reducing bacterium, S. putrefaciens is known to localize the majority of its membrane-bound cytochromes on its outer membrane (Myers and Myers, 1992, Pham et al., 2003). The cytochromes on the outer membrane are believed to be involved in the reduction of water soluble Fe(III). Intact cells of anaerobically grown S. putrefaciens were electrochemically active and the bacterium could grow in a fuel cell-type electrochemical cell in the absence of electron acceptors. Similar studies were made using another Fe(III) reducing bacterium G. sulfurreducens. Desufobulbus propionicus was found to grow with Fe(III) or humic acid analogue or a graphite electrode as electron acceptor (Holmes et al., 2004b). In these cases the electroactive enzymes present in the outer membrane of the cell is responsible for direct electron transfer between microorganisms and electrode. Dissimilatory ferric reduction is proposed to be an early form of microbial respiration. It has been shown (Varges et al., 1998) that Archaea and Bacteria, which are very closely related to the last common ancestor, can reduce Fe(III) to Fe(II) and generate energy from the respiration to support growth. Dissimilatory iron reducing microorganisms designated as GS-15, have been recently discovered that they reduce extra-cellular amorphous Fe(III)-oxyhydroxide as the terminal electron acceptor for the oxidation of organic matter to magnetic iron oxides under anaerobic conditions (Lovely et al., 1987, Pierre et al., 2002). This is an interesting finding since this microbial metabolism might have played an important role in the magnetization of anaerobic sediments and could account for the accumulation of magnetite in ancient iron formation. Magnetite accumulation has also been used as an indication that Fe(III) reduction was an important respiratory process on early Earth (Walker, 1987) and is significant in modern hot deep terrestrial biospheres, allowing a life in the absence of solar energy (Liu et al., 1997). So far only two classes of Ferricyanide reductases are known, the soluble prokaryotic Flavin reductase and the membrane cytochrome b like reductases found in prokaryotes. Ferricyanide reductase of Saccharomyces cerevisiae has been analysed in detail and it is characteristic of a b-type cytochrome and very similar to flavocytochrome b558 of human neurophils. This enzyme takes electrons from NADPH in the cytoplasm and passes them across the membrane via FAD and heme to molecular oxygen generating superoxide that is expelled into the lumen of the vacuole (Shatwell et al., 1996).

Electrochemical techniques have been extensively used to characterize redox proteins including cytochromes. In general, bacterial cells containing electrochemically active proteins tend to be electrochemically inactive as their cell wall structures consist of non-conducting material such as lipid and peptidoglycan (Park and Zeikus, 2002). Hence mediators were used to facilitate electron transfer between an electrode and electrochemically inactive microbial cells. Alternatively, the bacterial cells can be modified with hydrophobic conducting polymers to render them electrochemically active (Park et al., 1997). In this work we have explored the possibility of direct electrical communication between the yeast cells and electrode surface and demonstrated current generation in aerobic microorganisms for the first time.

The mediatorles systems described so far make use of Ferric ions as terminal electron acceptors instead of oxygen. During electron generation the reduction of ferric ions is found to be the intermediate step before electron collection on electrode surfaces. In the case of Geobacter sulfurreducens and D. propionicus the dissimilatory iron reduction is the intermediate step before electron generation at the electrode surface. In the case of S. putrefaciens, the cytochromes present in the outermembrane are involved in the reduction of water soluble ferric salts and this is the intermediate step before electron generation. In the present work, the redox proteins present in the outer membrane of the yeast cells facilitate direct electron transport between bacterial steps without any external mediators and without the intervention of reduction of ferric ions as the intermediate step. It is likely that the redox potentials of the proteins ferricyanide reductase and Lactate dehyrogenase and that of the electrode material (present in the membrane fraction of Hansenula anomala) are favorably arranged for the electron transport relay from the cells directly to the electrode surface. The efficiency of current generation has been evaluated using three anode materials.

Section snippets

Sub-culturing of H. anomala

H. anomala was sub-cultured using the medium comprising d-glucose: 1 g; peptone: 0.5 g; malt extract: 0.3 g; yeast extract: 0.3 g; phosphate buffer: 100 ml.

The buffer solution was prepared by dissolving 1 g of KCl; 6 g of NaH2PO4; 2.9 g of NaCl and 2 g of Na2CO3 in 1 l.

Construction of the biofuel cell

A two-compartment cell made of Perspex with Nafion (961) membrane as the separator was used. Three different anode materials were used for the investigations—graphite, graphite felt and polyaniline (PANI)–Pt composite coated graphite

Analysis of membrane fraction of H. anomala

The membrane fraction of the species H. anomala has been separated and analyzed. The presence of some redox enzymes could be demonstrated by our analysis. The isolated membrane fraction was found to contain Lactate dehydrogenase (cytochrome b2), NADH-Ferricyanide reductase, NADPH-Ferricyanide reductase, and cytochrome b5 (see Supplementary files for bar graph). The total protein content of the membrane fraction was found to be 52.35 μg protein/10 μl of the sample.

In earlier reports, the presence

Conclusions

In this work, H. anomala, which belongs to the family of yeast, is demonstrated to exhibit direct electron transfer without the aid of mediators. The electro-chemical activity of the microorganisms could be demonstrated by two different immobilization methods. The redox enzymes present in the cell membrane, viz., ferricyanide reductase and lactate dehydrogenase which are responsible for its electroactivity, have been analyzed by extracting the membrane fraction of the yeast cells. It has also

Acknowledgements

The authors wish to acknowledge Director, CECRI for his keen interest in this work Supplementary files available for schematic diagram representing the Type-I and Type-II electrode assemblies and bar graph representing the results of enzyme analysis in the membrane fraction.

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