The increasing concern about water quality and energy demand promotes the development of innovative and low-cost processes to improve the nutrient uptake and energy efficiency of existing wastewater treatments (WWT). In this context, the inclusion of a microalgae system (MAS) in the flowsheet of a WWT plant represents a sustainable alternative to conventional technologies, as it combines a low-cost nutrient uptake system with the production of biomass suitable for biofuel production. However, at present, the energy required to cultivate and process the algae cells is often too high to justify their use. The adoption of a low energy harvesting system and an efficient energy conversion process are the sine qua non requirements to guarantee the sustainability of the process. In this thesis, current and innovative harvesting technologies for large scale applications have been reviewed to identify the optimal working conditions of each system and their link to the main characteristics of the algae suspension. In particular, the performance of the Ballasted Dissolved Air Flotation (BDAF) system was investigated using different algae and compared to the conventional Dissolved Air Flotation (DAF). BDAF was demonstrably a very viable harvesting method where the use of floating microspheres as ballasting agents allowed significant coagulant savings, reduced the level of energy dissipation within the flotation chamber, and lowered the overall carbon emissions and the process costs. Cont/d.

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.