Two criteria that drive strategies for algae-based wastewater treatment for biofuel production are the need to produce a high-quality effluent on a consistent basis and the need to produce biomass with high oil content. While there are proponents for hydrothermal liquefaction (HTL) treatment of algal biomass to develop a “green crude,” the goal in the scenario described above is to separate the oil and the biomass. The nitrogen and phosphorus content captured in the biomass is needed in the production of methane in the digester, and later, the biosolids from the digester can be composted with green waste to make a high-quality fertilizer.

To meet both criteria, compromises must be made in the selection of algae. In traditional wastewater oxidation ponds, there are a wide array of different pro­karyotic and eukaryotic photosynthetic organisms. These ponds are subject to seasonal shifts in dominant populations as well as changes due to predation by protozoans and zooplankton. The variation in algal population dynamics can be minimized by periodic inoculation of the pond with a desired unialgal strain cul­tured in photobioreactors. Another consideration is the relationship between bac­teria in the wastewater and the algae. In addition, evidence exists for a role for heterotrophic microbes in algae auto-flocculation (Lundquist et al. 2011). A close examination of algae collected from wastewater ponds using a light microscope will usually show a significant number of heterotrophs associated or attached to the surface of the algae. If the algae are to be co-cultivated in photobioreactors as described above, it would be prudent to include the strains of wastewater bacteria associated with the specific type(s) of algae and which promote flocculation. The microalgae encompass a phylogenetically diverse assemblage of prokaryotic and eukaryotic photosynthetic microorganisms found in a wide range of habitats ranging from terrestrial environs to fresh and marine to hypersaline waters. It follows then that the ecology, morphology, biochemistry, and physiology are also diverse. Although the number of algal species is estimated to range between 30,000 and 300,000 with 7500 species systematically estimated from the literature (Guiry

2012) , less than 1 % have been isolated and characterized (Radner and Parker

1994) . Thus, the biotechnological potential of these microorganisms is just beginning to be explored for production of high-value and value-added products and biofuels. Microalgae have been used for decades as a source of high-value compounds with pharmaceutical activity including anticancer, antimicrobial, anti­viral agents, and pigments including a variety of carotenoids, cosmetics, nutra — ceuticals, and feed supplements for poultry, livestock, and mariculture (see Walker et al. 2005 for a review). Many groups are now exploring the use of transformable eukaryotic strains to produce heterologous proteins since they are capable of intron- splicing, glycosylation, and multimeric protein assembly (Spolaore et al. 2006).

Several aspects of algae biology and physiology are relevant to their economic potential of microalgae as a feedstock for biodiesel coupled to bioremediation. Of particular interest to the biofuels industry are productivity, biochemical composi­tion, and the influence of environmental and cultural practices on physiological processes, especially lipid metabolism and its regulation, photosynthetic efficiency, cell wall structure, and heterotrophic/mixotrophic capabilities. In addition, tech­niques to control algal/microbial pond community structure are essential for quality control of biodiesel composition from algae biomass. The ASTM International consensus-based standards group, whose standards are recognized in the United States, have specifications for the quality of biodiesel. The fuel characteristics are strongly influenced by FAME composition including chain length and degree of saturation. FAMEs isolated from algae species range in size from 12 to 38 carbons. The hydrocarbons that comprise petroleum products range in length as follows: 5-12 carbons for gasoline, 10-15 for diesel fuel, and 12-16 for kerosene (the main component of jet fuel). Refineries crack the longer hydrocarbons found in crude oil, then distill and blend the resulting compounds to formulate standard petroleum products. Maintenance of species composition, especially in outdoor ponds and when using wastewaters, is problematic. Cultivation of Spirulina in open outdoor ponds has been a success story in commercial algaculture. This strain grows in nearly pure culture in the alkaline, high-salinity waters of Lago de Texcoco. Competition from invading species is minimized due to the inhospitable nature of these waters. Control of species composition is crucial to quality control of biodiesel production. Lipid profiles are characteristic of some organisms and have even been used as a taxonomic feature. However, microalgae show great inter — and intraspecific variation in fatty acid profiles and these profiles can be affected by culture conditions (Roessler 1990).