This Small Business Innovation Phase I project will develop enzyme products from algae along with lipid biofuels. Algae have the potential to provide carbon-neutral, renewable biofuels from advanced lipids. At most, algae will be capable of producing advanced lipids as 30% - 40% of their total biomass, leaving 60% - 70% of the remaining biomass, primarily protein, as waste. As algae growth for biofuel production is scaled up to the levels required to replace significant amounts of transportation fuel, the costs for disposing of this waste biomass will become prohibitive. Therefore, identifying and developing large-scale commercial uses for the remaining biomass is required for commercial viability of large scale advanced lipid biofuel production from algae. In order to fulfill this need, Sapphire Energy will develop a system to co-produce commercially important industrial enzymes and advanced lipids for biofuels in algae. Stephen Mayfield of Sapphire Energy has pioneered the recent development of tools to overexpress proteins in algae. The Phase I experiments examine the feasibility of using these recently developed tools to produce industrial enzymes in algae at levels sufficient to be competitive with existing enzyme production systems while retaining activity and purity. The broader impacts/commercial potential of this project is to make algae more viable as an alternative fuel while also supplying industrial enzymes. Revenue from the sale of industrial enzymes will negate the costs of disposal of excess protein byproduct resulting from lipid production in algae, lowering production costs of advanced lipid biofuels. Development of this technology has wide-ranging societal impacts as it would reduce a key barrier to commercialization of a renewable transportation fuel that 1) can directly substitute for petroleum-based gasoline in current automobile engines, 2) does not compete with or use food crops, and 3) utilizes wastewater and excess CO2. Additionally, the work will allow further development of molecular tools for manipulating algae, increasing the types of products that may be produced in an organism that is rapidly gaining importance for the production of carbon-neutral, renewable products