Engineering

II. B.3.a. Introduction

The overall goal of the ASP was to cost-effectively produce biodiesel fuel from microalgal lipids. The early laboratory efforts focused on the characterization of microalgae with regard to traits deemed desirable for mass culture and fuel production, i. e., rapid growth, tolerance to environmental fluxes, and high production of TAG storage lipids. Although numerous promising organisms were identified, no individual strain demonstrated rapid growth with constitutively high lipid production. Although high lipid levels could be induced in many strains by starving the cells for an essential nutrient such as N or Si, the increase in lipid was accompanied by a decrease in cell division and total productivity.

During the late 1980s and 1990s, the direction of the laboratory research efforts at NREL shifted to the study of the biochemical pathways involved in lipid synthesis, with the goal of identifying targets for genetic manipulation. As discussed earlier, the desirable traits for biodiesel production (high productivity and high lipid content) were found to be mutually exclusive conditions in the organisms studied. Therefore, it was decided to use mutagenesis or genetic engineering to manipulate the algal biosynthetic pathways to produce algal strains with constitutively high lipid levels. Another possibility would be to engineer an organism in which lipid synthesis could be regulated by inducing or repressing key genes. Very little was known about the molecular biology of oleaginous microalgae and the genetic regulation of lipid biosynthesis pathways, so a concentrated research effort in this direction was deemed critical to

the success of the biodiesel project. Another reason for the shift to research on genetic manipulation of algae was more practical. Funding levels for ASP decreased during this period from the high funding levels in the mid-1980s that had allowed large numbers of subcontractors and the development of the Outdoor Test Facility in Roswell, New Mexico (Section II. B.5.). Laboratory experiments emphasizing biochemistry, molecular biology, and genetic engineering could be performed with a limited budget and few personnel.

This section of the report will describe the in-house research efforts at NREL to develop high lipid algae by genetically manipulating selected oleaginous strains. Microalgae generally reproduce asexually by simple fission. Many strains can also produce sexually, but the conditions required to induce algal sexual reproduction in the laboratory are not known for most species. Thus, genetic manipulation by classical “breeding” was not an option for the algae. The approaches explored at NREL were (1) mutagenesis and selection; and (2) genetic engineering. The information summarized in this report was taken from annual reports, scientific publications and meeting reports. No annual reports were generated by the ASP after 1993, and no quarterly reports after September 1995, so some of the most recent information presented was derived from the personal experience of Terri Dunahay and discussions with former coworkers (Paul Roessler and Eric Jarvis).