Microalgae produce large quantities of lipids that can be used for biofuel feedstock. The goal of this project was to determine the effect of several engineering and management parameters on the productivity of microalgae cultivated in large, outdoor facilities. The specific objectives were focused on the effects of inoculation ratios; the effects of light, temperature, and culture depth on growth; and the minimum circulation velocity necessary to maintain growth and minimize settling in open ponds. Microalgae must first be cultured in smaller quantities before the raceway is inoculated for optimized growth. Concentration ratios are defined as the ratio of the volume of microalgae inoculum to the volume of new growth media. The microalgae species used was Nannochloris oculata (UTEX #LB 1998). Inoculation ratios studied varied from 1:1 to 1:32 and were grown in 500 mL Erlenmeyer flasks. The study found that 1:16 and 1:32 were too dilute, while the 1:8 concentration had the largest growth rate. Determination of the effects of temperature, light intensity, and cultivation depth is critical to maintaining healthy cultures. Excess light intensity can result in photoinhibition and temperatures above the maximum growing tolerance can have detrimental effects. These factors can affect growth and evidence suggests an interaction that exacerbates these effects. In an outdoor culture there are few practical control variables other than pond depth. As cultivation depth increases, the algae undergo "selfshading" and the increased cultivation volume hinders temperature changes. Scaled raceway ponds were maintained at 10.16 cm (4 inch) and 13.97 cm (5.5 inch) depths. The species used was Nannochloris oculata and it was found to grow best at 785 micromol m⁻² s⁻¹m^-2 s^-1, 20°C, and 10.16 cm. Diatoms are attractive because of high growth rates, faster lipid production, and greater cell density. The latter promotes rapid settling once mixing has stopped. Because of the silica cell wall composition, diatoms are believed to be more susceptible to shear forces which can result in lysis. Determining the natural settling rate to the minimum channel velocity relationship in cultivation ponds was the objective. No flocculants/coagulants were added which created a case of "natural" settling. Four pennate Nitzschia sp. and one centric diatom were tested in a jar tester. There was no significant difference in settling times between the species. The mean settling time was 4.55 cm min⁻¹ and the minimum channel velocity was determined to be 10.12 cm min⁻¹.

This dissertation investigates harvesting of marine microalgae for bioenergy and production of two recombinant proteins for therapeutic applications in Chlamydomonas reinhardtii. The first study describes harvesting of marine microalgae by flocculation using aluminum chloride (AlCl_3), natural polymer chitosan, and synthetic cationic polymers. Harvesting and concentration process of low concentration microalgae cultures ranging from 1 to 2 g dry weight per liter was affected by algogenic organic matter (AOM), ionic strength, cell concentration, polymer charge density, and media pH. Marine microalgae flocculation was greatly affected by the presence of AOM independently of the flocculant chemistry. Presence of AOM demanded extra flocculant dosage i.e., 3-fold of AlCl3, 7-fold of highly charged synthetic cationic polymer, and 10-fold of chitosan. Flocculant dosage required for > 90 % flocculation efficiency in the presence of AOM was 160 mg/L, 50 mg/L, and 20 mg/L when using AlCl_3, chitosan, and best (more efficient) synthetic polymer respectively. The high-ionic strength of saline water did not have a significant effect on flocculation efficiency when using AlCl_3. However, to achieve efficient algal biomass removal, application of highly-charged synthetic polymers was required to overcome the presence of electrolytes. The best synthetic cationic polymer tested herein, which achieved greater than 90 % flocculation efficiency at 20 mg/L dosage, was a polymer with 99 % cationic charge density. Cell concentration also affected flocculant dosage requirement; low density cultures (10^6 cells/mL) required 6-fold greater dosages than cultures grown until early stationary phase (10^7 cells/mL). The second study addresses cultivation, extraction and purification challenges of two complex recombinant proteins, an immunotoxin molecule (MT51) and malaria vaccine antigen (Pfs25) produced in the chloroplast of C. reinhardtii. Main challenges identified were i) low transgene expression level, ii) proteolytic instability of MT51 immunotoxin, and iii) aggregation of Pfs25 antigen. Optimal expression and accumulation of Pfs25 antigen required growing C. reinhardtii cultures to late exponential phase (10^6 cells/mL) and inducing transgene expression for 24 h at a photon irradiance of 120 µmol/m^2s.