SBIR-STTR Award

Algae are fast becoming recognized as superior to land plants for future sources of sustainable, high energy-content fuels, and as CO2 mitigation vehicles. A major bottleneck in developing algae farming as a "cleantech" industry is lack of quality seedstock for the algae farmer-growers. Live algae concentrates are highly perishable and simply unavailable in the trade. This project provides a means for high-performance algae to be stabilized at ambient temperatures as a viable cell concentrate for inventory storage and global shipping purposes. This novel solution permits a reliable route to seeding of algae farms by contract manufacturers producing algae biomass and rapid replacement of cultures contaminated during production in the field. It also provides a means for enhanced stock culture by algae stock centers. OBJECTIVES: The primary goals of this Phase I study are to stabilize and immobilize bioprocess algae at ambient temperatures as viable cell concentrates. This will be achieved through two technical objectives: 1) Establish functional recovery following forced quiescence of select bioprocess algae; and 2) Create a macro-encapsulation protocol for concentrated live algae. APPROACH: The approach for Objective 1 includes a) Comparative cryopreservation using best-in-hand protocols; b) Preservative loading as alternative to deep-freeze storage. Preservation of viable cells up to 6 months will be determined, with regular counts made. One-way analysis of variance (ANOVA) is performed using a level of significance set at 5%. Following quiescent storage, a cell activation step is carried out either by thawing or rehydration in culture medium. Viability is then assessed using iodine reagent or using bis-oxonol fluorescent dye coupled with laser flow cytometry; and c) Genetic analysis of non-conserved regions for rRNA genes using established protocols. The approach for Objective 2 is to form a three-dimensional biofilm of cross-linked fluids by macroencapsulation of algal concentrate using variations of sodium-alginate solution and cell densities. Cell growth calculations are plotted for specific durations of immobilization based on viable cell counts and analyzed by ANOVA

Microalgae have long been employed in basic research of plant genetics and physiology. Thus, continued research and development are essential to attain commercial viability of microalgae as a renewable source of industrial product feedstocks. This project addresses genetic solutions for algae as feedstock biofactories. OBJECTIVES: Specific goals of this project include the design and construction of chloroplast and nuclear genome vectors which will be used for the transformation of three species of commercially-relevant microalgae. A successful Phase II project will enable production of a protein with an immediate commercial market as a food additive. A secondary goal of this project will facilitate production of high levels of lipids in microalgae. The intent of this innovation research is to improve algal biotechnology and secure microalgae as a renewable rapidly-upscaled bioproduction crop with broad commercial applications. APPROACH: In the Phase I research, we were successful in developing novel chloroplast vectors and transformation methods in Dunaliella. Phase II will expand the scope of the work to include additional microalgae species with a demonstrated utility in large scale commercial cultivation. This research will design and construct strain-specific chloroplast and nuclear vectors to broaden the capabilities of algal biotechnology and allow a wide range of commercial goals with high productivity. This process will include the development of both constitutive and inducible promoters for enhanced expression of the genes of interest. Effective promoters must demonstrate enhanced reporter gene expression over non-transformed controls. Success of algal transformation will be measured in vivo by detection of fluorescence or resistance to the antibiotics used for selection