Abstract
In microbial electrochemical systems, transport of electrons from bacteria to an electrode is the key to its functioning. However, the roles of several electron transport proteins, especially the membrane-bound dehydrogenases which link cellular metabolism to EET pathway are yet to be identified. NDH-2 is a non-proton pumping NADH dehydrogenase located in the inner membrane of several bacteria like Bacillus subtilis, Escherichia coli, etc. Unlike NADH dehydrogenase I, NDH-2 is not impeded by a high proton motive force thus helping in the increase of metabolic flux and carbon utilization. In the current study, NADH dehydrogenase II protein (NDH-2) was heterologously expressed from B. subtilis into E. coli BL21 (DE3) for enhancing electron flux through EET pathway and to understand its role in bioelectrogenesis. We found that E. coli expressing NDH-2 has increased the electron flux through EET and has shown a ninefold increase in current (4.7 μA) production when compared to wild strain with empty vector (0.52 μA). Furthermore, expression of NDH-2 also resulted in increased biofilm formation which can be corroborated with the decrease in charge transfer resistance of NDH-2 strain and increased NADH oxidation. It was also found that NDH-2 strain can reduce ferric citrate at a higher rate than wild type strain suggesting increased electron flux through electron transport chain due to NADH dehydrogenase II activity. Purified NDH-2 was found to be ∼42 kDa and has FAD as a cofactor. This work demonstrates that the primary dehydrogenases like NADH dehydrogenases can be overexpressed to increase the electron flux in EET pathway which can further enhance the microbial fuel cells performance.