Title: Development of Electrochemical Methods for Detection of Pesticides and Biofuel Production
Description: Electrochemical methods coupled with biological elements are used in industrial, medical and environmental applications. In this thesis we will discuss two such applications: an electrochemical biosensor for detection of pesticides and biofuel generation using electrochemical methods coupled with microorganisms. Electrochemical biosensors are commonly used as a result of their selectivity, sensitivity, rapid response and portability. A common application for electrochemical biosensors is detection of pesticides and toxins in water samples. In this thesis, we will focus on detection of organosphosphates (OPs), a group of compounds that are commonly used as pesticides and nerve agents. Rapid and sensitive detection of these compounds has been an area of active research due to their high toxicity. Amperometric and potentiometric electrochemical biosensors that use organophosphorus hydrolase (OPH), an enzyme that can hydrolyze a broad class of OPs, have been reported for field detection of OPs.

Amperometry is used for detection of electroactive leaving groups and potentiometry is used for detection of pH changes that take place during the hydrolysis reaction. Both these methods have limitations: using amperometric biosensors, very low limits of detection are achieved but this method is limited to the few OPs with electroactive leaving groups, on the other hand potentiometric biosensor can be used for detection of all OPs but they don't have low enough limits of detection. We have developed a novel dual enzyme biosensor biosensor with OPH and horseradish peroxidase (HRP) for detection of OPs with phenolic leaving groups which are much more common than electroactive leaving groups. This biosensor was used for detection of dichlofenthion, an OP which does not have an electroactive leaving group. Electrochemical biosensors with enzymatic layers such as the one described above are commonly used, however there are no clear guidelines on how to design these sensors. Based on the application, the biosensor might be designed for a target limit of detection, sensitivity and/or linear range.

Simulation tools can be used to provide general guidelines that can be applied to a wide range of biosensors and reduce extensive development time. In chapter 3, reaction-diffusion equations for an enzymatic biosensor with saturation kinetics are solved. Dimensionless numbers that can guide the researchers on designing and optimizing biosensors are presented. Additionally results from a formaldehyde biosensor are presented to demonstrate the use of some of the guidelines introduced. Recently biofuel production using electricity has become an attractive area of research in the interface between electrochemistry and biology. Biofuel production from renewable resources is the challenge of our century. Bioproduction using natural photosynthesis routes suffers from low efficiencies. An alternative to photosynthesis is the use of chemolithoautotrophic organisms that are genetically engineered to produce biofuels. These organisms use inorganic redox mediators as their energy source instead of photosynthesis.

A sustainable biofuel production process is feasible, if the chemolithoautotrophic organisms are coupled with an electrochemical reactor to recycle the redox mediator. We will describe the development of an electrochemical reactor that reduces nitrite to ammonia which is the natural mediator couple for the chemolithoautotrophic organism Nitrosomonas europaea. Using nickel and glassy carbon electrodes nearly 100% current efficiencies were obtained in batch electrolysis experiments in media optimal for N.europaea growth. Additionally effect of production of the potential biofuel isobutanol on cathode performance was studied. A flow-by electrochemical reactor with a porous electrode was designed to supply sufficient ammonia to a 5 L chemostat containing wild type N.europaea. Biomass production using electrochemically regenerated mediator was demonstrated by coupling this electrochemical reactor with N.europaea chemostat. Ammonia removal and biomass yield for the bacteria were unaffected by the coupled operation which suggests the electrochemically reduced media did not alter aerobic metabolism of the bacteria.

Reduction of nitrite to ammonia was further investigated using linear sweep voltammetry and electrochemical impedance spectroscopy. A limiting current plateau was identified in linear sweep experiments. In the pH range between 6.0 and 8.0, the flux of protons to the electrode were determined to cause mass transfer limitations. The same electrochemical methods were also used to assess the changes in performance of cathodes with time. Formation of a film was observed on glassy carbon and nickel electrodes. The film did not seem to cause any changes to the nickel electrode. On the other hand overpotential for the glassy carbon electrode was significantly reduced which would decrease power requirements for the electrochemical reactor and the overall process.