Current sources of fermentation feedstocks, i.e. corn, sugar cane, or plant biomass, fall short of demand for liquid transportation fuels and commodity chemicals in the United States. Aquatic phototrophs including cyanobacteria have the potential to supplement the supply of current fermentable feedstocks. In this strategy, cells are engineered to accumulate storage molecules including glycogen, cellulose, and/or lipid oils that can be extracted from harvested biomass and fed to heterotrophic organisms engineered to produce desired chemical products. In this manuscript, we examine the production of glycogen in the model cyanobacteria, . strain PCC 7002, and subsequent conversion of cyanobacterial biomass by an engineered to octanoic acid as a model product. In effort to maximize glycogen production, we explored the deletion of catabolic enzymes and overexpression of GlgC, an enzyme that catalyzes the first committed step towards glycogen synthesis. We found that deletion of increased final glycogen titers when cells were grown in diurnal light. Overexpression of GlgC led to a temporal increase in glycogen content but not in an overall increase in final titer or content. The best strains were grown, harvested, and used to formulate media for growth of . The cyanobacterial media was able to support the growth of an engineered and produce octanoic acid at the same titer as common laboratory media.

MR-1 is a dissimilatory metal reducing bacterium with a highly branched respiratory electron transport chain. The MR-1 genome encodes four NADH dehydrogenases, any of which may be used during respiration. We previously determined that a double-knockout of two NADH dehydrogenases, Nuo and Nqr1, eliminated aerobic growth in minimal medium. However, the double-knockout strain was able to grow aerobically in rich medium. Here, we determined that amino acid supplementation rescued growth of the mutant strain in oxic minimal medium. To determine the mechanism of the growth defect, we monitored growth, metabolism, and total NAD(H) pools in MR-1 and the NADH dehydrogenase knockout strain. We also used a genetically encoded redox sensing system and determined that NADH/NAD was higher in the mutant strain than in the wild-type. We observed that the double-knockout strain was able to metabolize d,l-lactate and N-acetylglucosamine when supplemented with tryptone, but excreted high concentrations of pyruvate and acetate. The requirement for amino acid supplementation, combined with an apparent inability of the mutant strain to oxidize pyruvate or acetate suggests that TCA cycle activity was inhibited in the mutant strain by a high NADH/NAD.

One of the challenges in Microbial Fuel Cell (MFC) technology is the improvement of the power output and the lowering of the cost required to scale up the system to reach usable energy levels for real life applications. This can be achieved by stacking multiple MFC units in modules and using cost effective ceramic as a membrane/chassis for the reactor architecture. The main aim of this work is to increase the power output efficiency of the ceramic based MFCs by compacting the design and exploring the ceramic support as the building block for small scale modular multi-unit systems. The comparison of the power output showed that the small reactors outperform the large MFCs by improving the power density reaching up to 20.4 W/m (mean value) and 25.7 W/m (maximum). This can be related to the increased surface-area-to-volume ratio of the ceramic membrane and a decreased electrode distance. The power performance was also influenced by the type and thickness of the ceramic separator as well as the total surface area of the anode electrode. The study showed that the larger anode electrode area gives an increased power output. The miniaturized design implemented in 560-units MFC stack showed an output up to 245 mW of power and increased power density. Such strategy would allow to utilize the energy locked in urine more efficiently, making MFCs more applicable in industrial and municipal wastewater treatment facilities, and scale-up-ready for real world implementation.

Proteins from the ferredoxin (Fd) and flavodoxin (Fld) families function as low potential electrical transfer hubs in cells, at times mediating electron transfer between overlapping sets of oxidoreductases. To better understand protein electron carrier (PEC) use across the domains of life, we evaluated the distribution of genes encoding [4Fe-4S] Fd, [2Fe-2S] Fd, and Fld electron carriers in over 7,000 organisms. Our analysis targeted genes encoding small PEC genes encoding proteins having ≤200 residues. We find that the average number of small PEC genes per Archaea (~13), Bacteria (~8), and Eukarya (~3) genome varies, with some organisms containing as many as 54 total PEC genes. Organisms fall into three groups, including those lacking genes encoding low potential PECs (3%), specialists with a single PEC gene type (20%), and generalists that utilize multiple PEC types (77%). Mapping PEC gene usage onto an evolutionary tree highlights the prevalence of [4Fe-4S] Fds in ancient organisms that are deeply rooted, the expansion of [2Fe-2S] Fds with the advent of photosynthesis and a concomitant decrease in [4Fe-4S] Fds, and the expansion of Flds in organisms that inhabit low-iron host environments. Surprisingly, [4Fe-4S] Fds present a similar abundance in aerobes as [2Fe-2S] Fds. This bioinformatic study highlights understudied PECs whose structure, stability, and partner specificity should be further characterized.