J. Joshi et al.: Optimization of External Potential for Ethanol Production by yeasts in electrochemical Cell Rentech Symposium Compendium, Volume 4, September 2014 44 Optimization of External Potential for Ethanol Production by yeasts in electrochemical Cell Jarina Joshi *1 , Rejeena Shrestha 1 , Rojlina Manandhar 1 , Dinita Sharma 1 , Krishna Manandhar 2 , Lakshmaiah Sreerama 3 , Doo Hyun Park 4 , Tribikram Bhattarai 1 1 Central Department of Biotechnology, Tribhuvan University, Nepal 2 Central Department of Chemistry, Tribhuvan University, Nepal 3 Department of Chemistry, St. Cloud State University, USA 4 Syokyong University, Seoul, Korea. Abstract - Ethanol was produced in electrochemical with combination of Saccharomyces cerevisiae and Wikerhamomyces anomalous . A combination of electrochemical cell with Saccharomyces cerevisiae grown in anode and Wikerhamomyces anomalous grown in cathode gave better ethanol production. Electrochemical cell with this combination of yeast was studied at different external voltage supply to see the effect in ethanol production. A supply of 4V was found to be best for ethanol production with total ethanol production of 379.76±9.52 mg/gm of glucose. When Platinum wire in anode coated with Platinum fine powder (Sigma) along with Nafion membrane in place of cellulose acetate was used, a drastic decrease in overall ethanol production was observed. The ethanol production was 138.68±1.54 mg/gm of glucose in anode and 243.85±34.76 mg/gm of glucose in cathode. Index Terms - Electrochemical Cell, Ethanol, External Voltage I. INTRODUCTION An electrochemical cell behaves like the whole cell metabolism on which oxidation and reduction occurs due to transfer of electrons through external circuit. As compared to the use of prokaryotes in the electrolytic cell, much less research have been carried out on the use of eukaryotes such as yeasts as biocatalysts in the electrolytic cell. Yeasts such as Saccharomyces cerevisiae and Candida melibiosica have the electrochemical capabilities to use as biocatalysts in such electrolytic cells (Hubenova.Y and Mitov .M, 2008). S. cerevisiae and few other yeasts are found to enhance ethanol production when grown in cathode with external voltage supply [Shin et al, 2002]. Some bacteria like Geobacteraceae sulferreducens, Geobacter metallireducens, Shewanella putrefaciens (Kim et al, 2010) and Rhodoferax ferrireducens (Chaudhari and Lovely, 2003) are known to form film on the anode surface and transfer electrons directly to the electrode across the membrane. Most microorganisms cannot transfer electrons directly to the electrodes. Neutral red, methylene blue, thionine, iron (III) EDTA, Meldola's blue, Mn 4+ as synthetic mediators and humic acids, Anthraquinone, the oxyanions of sulphur (sulphate and thiosulphate) as endogenous mediators (Park et al, 2000) are commonly used as external electron transport mediators (Hahn-Hägerdal et al., 2006) efficiently when external voltage is supplied. However very few study was conducted to see the effect on eukaryotic cells. This paper studied few possible parameters for optimized production of ethanol from yeasts in electrochemical cell. * Corresponding author: jarinarjoshi@gmail.com II. MATERIALS AND METHODS All the chemicals were purchased from HiMedia except few stated chemicals. Yeast strains Saccharomyces cereviseae (CDBT2) and Wikerhamomyces anomalous (CDBT7) were used as yeast strains. The yeasts were collected from culture collection of Central Department of Biotechnology, Tribhuvan University, Nepal. Yeast Maltose Agar (YMA) media with composition Yeast extract (3 gm/l), Malt extract (3 gm/l), Peptone (5 gm/l) and Glucose (10 gm/l), pH 4.5 were used for revival of culture from glycerol stock. [Middelhoven et al 1998; Middelhoven et al 2002]. Ethanol concentration was measured after solvent extraction with tri- n-butyl phosphate and treating with acidified potassium dichromate as described by Seo et al, 2009. 2.1 Ethanol Production in Electrochemical Cell 2.1.1 Immobilization of Neutral red to Graphite Electrode Porous graphite felt initially soaked in methanol was dipped in 1% polyvinyl alcohol solution for 3 to 4 hours. It was then dried in oven at about 80 o C for 24 hours. Completely dried graphite felt was then soaked in pure chloroform containing 10% thionylchloride and 0.01% neutral red (Sigma Co.) for 6 hours. Graphite felt was then left for overnight for air dry. It was then autoclaved and washed in running water till color persist. Finally, it was dried at 60 o C for 24 hrs and used as NR immobilized graphite electrode (Jeon et al, 2010). 2.2 Electrochemical Cell construction A two compartment electrolytic cell was designed to induce the electrochemical reaction. The anode and cathode compartments were initially separated by cellulose acetate film guarded by whatmann no.1 filter paper. Anode is made from Platinum electrode (0.2 mm diameter, Sigma) and cathode is made from neutral red immobilized graphite fiber. Working volume of the electrolytic compartments were adjusted to 60 ml each. 2.3 Ethanol Production in Electrochemical Cell Saccharomyces cerevisiae and Wikcerhamomyces anomalous were cultivated with either combination in anode and cathode alternatively. Once S. cerevisiae in anode and W. anomalous in cathode then vice versa and see the effect on ethanol production.