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Microbial natural products exhibit a wide range of ecological functions including antibacterial, iron scavenging, and quorum sensing capabilities. However, these molecules can also play tangential roles in primary metabolism. Bacterial respiration occurs in many different forms, often subject to the availability of terminal electron acceptors such as oxygen. However, when terminal electron acceptors become scarce, the electron transport chain locks up and energy production stops. Interestingly, some bacteria have evolved methods of extending their central metabolism outside of the cell in order to access distant terminal electron acceptors, stabilizing their electron transport chain. One observed method of extracellular electron transport involves the secretion of redox active small molecule electron shuttles, which are capable of extracellularly extending the cells redox capabilities. These natural products play a crucial physiological role that allows aerobic bacteria to survive in oxygen limited environments. This thesis explores our current understanding of this relatively underexplored function of small molecule natural products, with a focus on how they may play a role in marine sediments. Moreover, information on their environmental distribution, biosynthesis, phylogeny, physiological roles, and biotechnological applications is also provided.

Microbial natural products exhibit a wide range of ecological functions including antibacterial, iron scavenging, and quorum sensing capabilities. However, these molecules can also play tangential roles in primary metabolism. Bacterial respiration occurs in many different forms, often subject to the availability of terminal electron acceptors such as oxygen. However, when terminal electron acceptors become scarce, the electron transport chain locks up and energy production stops. Interestingly, some bacteria have evolved methods of extending their central metabolism outside of the cell in order to access distant terminal electron acceptors, stabilizing their electron transport chain. One observed method of extracellular electron transport involves the secretion of redox active small molecule electron shuttles, which are capable of extracellularly extending the cells redox capabilities. These natural products play a crucial physiological role that allows aerobic bacteria to survive in oxygen limited environments. This thesis explores our current understanding of this relatively underexplored function of small molecule natural products, with a focus on how they may play a role in marine sediments. Moreover, information on their environmental distribution, biosynthesis, phylogeny, physiological roles, and biotechnological applications is also provided.

Soil and groundwater pollution by chlorinated aliphatic hydrocarbons (CAHs) poses a threat to environmental and human health. A large number of sites around the world are contaminated with the CAHs carbon tetrachloride (CT) and perchloroethene (PCE). The employment of bacteria to remediate pollution events, termed in situ bioremediation, is currently gaining momentum as an effective and environmentally friendly treatment technology. A class of anaerobic bacteria named organohalide respiring bacteria (ORB) are able to remove PCE directly by utilizing it as an electron acceptor for growth. Unfortunately, no such bacteria are known which can transform CT in the same manner. Furthermore, CT and its partly dechlorinated product chloroform (CF) negatively impact the enzymatic degradation of PCE by ORB, creating major challenges for the bioremediation of sites co-contaminated with CT and PCE.While it cannot be used as a growth substrate, CT can be transformed by certain reactive metabolites of anaerobic bacteria. In particular, sulphide (HS-) and ferrous iron (Fe(II)) generated by sulphate- and iron-reducing bacteria (SRB and IRB) can transform CT. In this work, experimental results are presented which support the exploitation of this CT transformation pathway combined with the activity of organohalide respiring bacteria in view of bioremediating mixed CT and PCE plumes. Firstly, experiments were carried out to investigate the solvent tolerance of anaerobic bacteria in general, and revealed that fermentative strains are more tolerant to CAHs than SRB and IRB. Avenues to take advantage of this finding are proposed. Secondly, IRB and SRB enriched from several environmental sources displayed the ability to survive and produce Fe(II) and HS- respectively in the presence of CT and PCE. Thirdly, the potential of naturally-occuring electron-shuttling compounds was tested for their enhancing effects on CT transformation by Fe(II) and HS-. Phenolic-rich solutions including tea and

Biogeochemical transformation is defined as "processes where contaminants are degraded by abiotic reactions with naturally occurring and biogenically-formed minerals in the subsurface" (AFCEE et al., 2008). These biogenically-formed minerals are created as a result of biogeochemical reactions typically under anaerobic iron- and sulfate-reducing conditions. Biogenically-formed minerals are particularly important because they are continuously replenished in the subsurface and have high surface area and reactivity. In situ biogeochemical treatment systems are defined as systems that capitalize and/or enhance such natural processes via engineered reaction zones in the subsurface. Examples of this application include permeable reactive barriers (e.g., biowalls) and injection of organic carbon (e.g., soluble electron donors or vegetable oil emulsions) into a contaminated aquifer with the specific purpose of creating biogenically-formed minerals. The use of organic carbon-based permeable reactive barriers for treating chlorinated ethenes is an emerging technology that has shown its potential to be a cost-effective method of treatment (AFCEE, 2008). A carbon-based substrate such as plant mulch or vegetable oil is used to promote in situ biogeochemical transformation of chlorinated solvents such as trichloroethene (TCE) and tetrachloroethene (PCE) into benign end products such as carbon dioxide, chloride ion, and water.

Microbial fuel cells (MFCs) represent a promising approach for sustainable energy production as they generate electricity directly from metabolism of organic substrates without the need for catalysts. However, the mechanisms of electron transfer between microbes and electrodes, which could ultimately limit power extraction, remain controversial. Here we demonstrate optically transparent nanoelectrodes as a platform to investigate extracellular electron transfer in Shewanella oneidensis MR-1, where an array of nanoholes precludes or single window allows for direct microbeelectrode contacts. Following addition of cells, short-circuit current measurements showed similar amplitude and temporal response for both electrode configurations, while in situ optical imaging demonstrates that the measured currents were uncorrelated with the cell number on the electrodes. High-resolution imaging showed the presence of thin, 4- to 5-nm diameter filaments emanating from cell bodies, although these filaments do not appear correlated with current generation. Both types of electrodes yielded similar currents at longer times in dense cell layers and exhibited a rapid drop in current upon removal of diffusible mediators. Reintroduction of the original cell-free media yielded a rapid increase in current to ?80% of original level, whereas imaging showed that the positions of >70% of cells remained unchanged during solution exchange. Together, these measurements show that electron transfer occurs predominantly by mediated mechanism in this model system. Last, simultaneous measurements of current and cell positions showed that cell motility and electron transfer were inversely correlated. The ability to control and image cell/electrode interactions down to the single-cell level provide a powerful approach for advancing our fundamental understanding of MFCs., Sponsored in part by the Air Force Office of Scientific Research (AFOSR). Performed in collaboration with Harvard University, Cambridge, MA. Published in the Proceedings of the National Academy of Sciences (PNAS), Early Edition, p1-5, 2010.

The original scope of this project involved demonstration and validation of the use of electron shuttles compounds for the oxidative biodegradation of cis-1,2- dichloroethene (cis-DCE) and vinyl chloride (VC). As results from the treatability study using radiolabeled vinyl chloride were not encouraging, the project was cancelled. Since this project was not completed as planned, this final report does not the follow the standard Environmental Security Technology Certification Program (ESTCP) report format. This report presents the findings from field sampling and analysis and treatability testing that was conducted at both Dr. Derek Lovley s and Dr. Frank Chapelle s laboratories., Prepared in collaboration with Camp Dresser & McKee Inc., Annandale, VA.