Monitoring metabolic pathway alterations in Escherichia coli due to applied potentials in microbial electrochemical system.

Redox potential is one of the key regulators in determining the fate of the metabolic pathways of biocatalysts and of their associated product synthesis in microbial electrochemical systems. In the present study, the influence of applied potentials on fermentation products and metabolic flux was investigated using isolated E. coli HP3 as a model organism using pyruvate as a substrate. To provide insights into metabolic shifts, electro-fermentative (EF) systems were constructed and poised at both positive and negative redox potentials of 0.2 V, 0.4 V, 0.6 V and 0.8 V (vs Ag/AgCl) at the anode. The relative expression of genes encoding lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), pyruvate dehydrogenase (aceF), hydrogenase (hycE) and NADH: oxidoreductase (nuoB) enabled assessment of metabolic shifts in addition to cyclic voltammograms and short chain fatty acid profiling. Results showed that poised conditions had a significant effect on product formation and observed up-regulation of key enzymes involved in pyruvate metabolism in comparison to controls. More specifically, EF poised at -0.8 V and -0.2 V enhanced H2 production by 7.9 folds and 5.3 folds respectively, whilst at +0.8 V poised operation enhanced lactate production by 1.9 folds compared to the control. Concomitantly, the key genes involved in the pathway for H2 production viz., plfB, hycE and nuoB were all up-regulated significantly in a reactor poised at -0.8 V compared with other conditions. Similarly, transcripts for gene ldhA were up-regulated significantly in the system poised with +0.8 V. The study elucidated the role of redox potential on the product formation with respect to the relative expression of the genes encoding key enzymes in the metabolic pathway in correlation with bio-electrochemical characteristics.

Purification and Characterization of NDH-2 Protein and Elucidating Its Role in Extracellular Electron Transport and Bioelectrogenic Activity.

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.

Selective enrichment of electrogenic bacteria for fuel cell application: Enumerating microbial dynamics using MiSeq platform.

This study is intended to examine the effect of pretreatment on selective enrichment of electrogenic bacteria from mixed culture. It has been observed that the iodopropane and heat-shock pretreatments suppress the growth of non-exoelectrons, while selecting only a limited number of strains belonging to genera Xanthomonas, Pseudomonas and Prevotella while untreated control inoculum showed more diverse community comprising of both exoelectrogens and non-exoelectrogens. High power output was observed in iodopropane (180mW/m(2)) pretreated microbial fuel cell (MFC) compared to heat-shock pretreated MFC (128mW/m(2)) and untreated control (92mW/m(2)). Coulombic efficiency of iodopropane and heat-shock pretreated MFC was higher compared to untreated control MFC, while drop in pH and volatile fatty acids (VFA) production was less in iodopropane pretreated MFC signifying the shifts in bacterial community structure toward electrogenesis instead of fermentation. These results signify the role of iodopropane and heat pretreatments on enrichment of electrogenic bacteria for fuel cell application.

Polarized potential and electrode materials implication on electro-fermentative di-hydrogen production: Microbial assemblages and hydrogenase gene copy variation.

This study examined the changes in microbial diversity in response to different electrode materials viz., stainless steel mesh (SS) and graphite plate as anodes in two microbial electrolysis cell (MEC) each poised at 0.2V, 0.4V, 0.6V and 0.8V. Changes in microbiota prior to and after pretreatment along with microbiota enriched in response to various poised potentials with SS and graphite are monitored by 16S rRNA gene based DGGE profiling. Significant shifts in microbial community were noticed at all these experimental conditions. Correspondingly, the level of hydrogenase belonging to genera Bacillus, Pseudomonas, Rhodopseudomonas and Clostridium was studied by quantitative real time PCR (RT-PCR) at various applied potentials. DGGE based 16S rRNA gene profiling revealed enriched members belonging to phylum Firmicutes predominantly present at 0.8V in both MECs contributing to high hydrogen production. This study first time explored the growth behavior of mixed consortia in response to poised potentials and electrode materials.

Microbial catalyzed electrochemical systems: a bio-factory with multi-facet applications.

Microbial catalyzed electrochemical systems (MCES) have been intensively pursued in both basic and applied research as a futuristic and sustainable platform specifically in harnessing energy and generating value added bio-products. MCES have documented multiple/diverse applications which include microbial fuel cell (for harnessing bioelectricity), bioelectrochemical treatment system (waste remediation), bioelectrochemical system (bio-electrosynthesis of various value added products) and microbial electrolytic cell (H2 production at lower applied potential). Microorganisms function as biocatalyst in these fuel cell systems and the resulting electron flux from metabolism plays pivotal role in bio-electrogenesis. Exo-electron transfer machineries and strategies that regulate metabolic flux towards exo-electron transport were delineated. This review addresses the contemporary progress and advances made in MCES, focusing on its application towards value addition and waste remediation.