While recent studies have demonstrated direct photosynthetic production of biofuels via engineered cyanobacteria, biofuel yields from cyanobacteria remain at low levels. As with heterotrophic biofuel production, product toxicity is likely a limiting factor. Some mechanisms of toxicity may be similar to those studied in common heterotrophic hosts; however, the photosynthesis-dependent pathways for carbon fixation and energy production in cyanobacteria present unique targets for biofuel toxicity. This study investigates biofuel toxicity for three model cyanobacterial strains: Synechococcus elongatus PCC 7942, Synechocystis sp. PCC 6803, and Synechococcus sp. PCC 7002. While cyanobacterial biofuel tolerances were generally lower than that of heterotrophic hosts, the marine strain Synechococcus sp. PCC 7002 showed increased tolerance to short chain alcohols, and long-chain biofuel products, such as fatty alcohols, saturated free fatty acids, alkanes, and alkenes had minimal toxicity for all three cyanobacteria. Targeted mutants were generated to explore natural mechanisms of biofuel tolerance in cyanobacteria, such as cell membrane composition, reactive oxygen species degradation, and efflux pumps. These mutants confirmed the influence of cell membrane composition on cyanobacterial tolerance to short-chain alcohols. This study provides data to guide both biofuel product and cyanobacterial host selection and further identifies potential targets for improving biofuel tolerance in cyanobacteria.

Algal biofuels are a renewable energy source with the potential to replace conventional petroleum-based fuels, while simultaneously reducing greenhouse gas emissions. The economic feasibility of commercial algal fuel production, however, is limited by low productivity of the natural algal strains. The project described in this SAND report addresses this low algal productivity by genetically engineering cyanobacteria (i.e. blue-green algae) to produce free fatty acids as fuel precursors. The engineered strains were characterized using Sandia's unique imaging capabilities along with cutting-edge RNA-seq technology. These tools are applied to identify additional genetic targets for improving fuel production in cyanobacteria. This proofof-concept study demonstrates successful fuel production from engineered cyanobacteria, identifies potential limitations, and investigates several strategies to overcome these limitations. This project was funded from FY10-FY13 through the President Harry S. Truman Fellowship in National Security Science and Engineering, a program sponsored by the LDRD office at Sandia National Laboratories. 4 AcknowledgmentsThe authors of this SAND report acknowledge the support of management, particularly Anthony Martino, sponsor of Anne Ruffing's Truman Fellowship application; Eric Ackerman, Truman Fellow mentor; James Carney, Project Manager; Blake Simmons; and Ben Wu. We thank Michelle Raymer and Omar Garcia for their assistance with MCR analysis and operation of the hyperspectral confocal fluorescence microscope. We also acknowledge

Major scientific challenges hinder the success of an industrial-scale algal biofuels program. Four broad areas of R&D needs have been identified for economically viable, industrial-scale cultivation of algae: culture sustainability; system productivity; nutrient source scaling and sustainability; and water conservation, management, and recycling. Progress in each of these areas is limited by significant knowledge gaps in fundamental algal biology. This SAND report summarizes research conducted as part of an LDRD project (FY10-FY12) to address this shortcoming. We have developed a novel, multidisciplinary, multiscale approach utilizing Sandia's core expertise in bioanalytical spectroscopy, chemical imaging, remote sensing, genomics, and computational modeling in collaboration with researchers at University of New Mexico and Arizona State University to investigate the effects that dynamic abiotic and biotic stressors have on algal photosynthesis, growth, and lipid production. The discoveries will enable gains in productivity and sustainability that are critical for cost-effective, industrial-scale algal facilities. 5 ACKNOWLEDGMENTS