Approximately fifty species are utilized in biotechnology, mostly as biofeed. In these “traditional” species, manipulations of culture conditions (i. e., temperature, light, and nutrients) dramatically influence the yield of biomass. Long-term maintenance of algae may result in loss of algal vigor, resulting in “culture crashes” (Russell, 1974).

Cryopreservation of algae also contributes to death of cultures (Day and Harding, 2008). Such effects become obvious over long periods but are not evident in short periods—for example, the loss of B12 requirement in axenic cultures with long-term maintenance (Andersen, 2005). Continuous vegetative reproduction may lead to degeneration of cells, and such lost vigor can be restored by periodic sexual repro­duction or the addition of organic base (Andersen, 2005). Growing multiple species may provide insight into their competition for nutrients and into the reproductive capacity of their vegetative stages (Riegman et al., 1996).

Cultivation of microalgae under laboratory conditions in defined sterile media, controlled temperature, and light influences their cost. For autotrophic microalgae, more than thirty kinds of media are used (Andersen, 2005; Subba Rao, 2009) and some of the commercially available nutrient stocks such as f/2 medium cost $25 per liter. However, for outdoor mass cultivation systems and commercial developments, less-expensive media based on the enrichment of wastewater should be preferred. It would be necessary to carry out pilot experiments to critically evaluate the suitability of these media because of variations in their chemical composition. The use of wastewater, eutrophified water (Woertz et al., 2009; Kong et al., 2010, Park et al., 2011), secondary sewage (Orpez et al., 2009), dairy manure (Wang et al., 2010), swine manure effluent (Kebede-Westhead et al., 2006), farm effluents (Craggs et al., 2004), and commercial fertilizers such as Clewat-32™ (Ronquillo et al., 1997), Nualgi, SB07321(LM)M, Dyna-Gro™, and Miracle-Gro® may substantially reduce these costs while promoting vigorous algal growth.

Several designs for large-scale, flat-bed plane photobioreactors (PBRs) are available. Algae are also grown in a closed-loop, vertical or horizontal system of polyethylene sleeves, known as high density vertical growth (HDVG) systems, in greenhouses (Ugwu et al., 2008). Where land is at a premium, as at Schiphol Airport, Holland, it is proposed to construct an “ecobarrier”—a long tent parallel to the runway (Natural Resources Defense Council, 2009). Although a futuristic speculation, it is hoped that the ecobarrier supports algal cultivation and biofermen­tation technologies, and integrates transportation and landscape (Natural Resources Defense Council, 2009). However, an evaluation of the impact of temperature and light on their performance to sustain biomass levels is difficult because of the natural conditions. Because flat-plate photobioreactors suffer from a lack of uniform avail­ability of light energy, it is suggested that circular-geometry bioreactors are better suited. Grobbelaar (2009) recommended closed PBRs because of their higher light utilization efficiencies, nutrient uptake, and biomass yield, and lower compensa­tion light:dark ratios or respiratory losses, less contamination, and less water loss. A mean maximum of 98 g m-2d-1 with a maximum productivity of 170 g m-2d-1 is claimed by the Green Fuel reactor (Pulz, 2007), which can be attributed to a high surface volume ratio (SVR). For industrial purposes, algae are mass cultivated by the Israel-based Seambiotic in open-pond raceways ranging from 200 L to 1.2 x 106 L covering a 3,400-m2 area.

Cultivation of algae under natural light and temperature is more cost-effective than under controlled laboratory conditions. Chlamydomonas sp., Chlorella sorokiniana, Dunaliella tertiolecta, D. salina, Haematococcus pluvialis, Nannochloropsis sp., Phaeodactylum tricornutum, Porphyridium purpureum,

P. cruentum, Scenedesmus obliquus, and Synechocystis aquatilis have been grown autotrophically in PBRs. Calculations with Dunaliella cultures showed that the use of a large, dense inoculum accelerates cell division with early attainment of stationary phase (Subba Rao, 2009). Such a shift saves time, which is desirable in a production process. Without negatively impacting growth rates, it is possible to attain a twofold increase in biomass in Neochloris oleoabundans by sequential increases in irradiance levels (Wahal and Vjamajala, 2010). Based on the geom­etry, fluid flow, and illumination on the biomass growth, Wu and Merchuk (2004) developed a triangular airlift reactor in which removal of CO2 by two green algae (Dunaliella parva and D. tertiolecta) in a pilot-scale unit supplied with flue gases from a small power plant was 82.3 ± 12.5% on sunny days and 50.1 ± 6.5% on cloudy days.

The University of California, San Diego, designed a multi-stage algal bioreactor at the Scripps Institution of Oceanography (http://techtransfer. universityofcalifornia. edu/NCD/21141.html). This reactor provides light-limited growth, different or com­bined nutrient-controlled regimes, and can pre-amplify algal production to continu­ously inoculate existing pond or bioreactor systems.

Ben-Amoz (2009) reported that aeration of mass cultures with CO2 flue gases enhances Dunaliella production from 2 to 20 g C m-2d-1 and could serve as an ideal and inexpensive nutrient source in commercial settings. Recent developments have substantially reduced production costs of microalgal dry biomass from $1,000 kg-1 in 1953 to a fraction of this ($0.17 to $0.29 kg-1) when grown in wastewater (De Pauw et al., 1984). Israel-based Seambiotic produces Dunaliella for $17 kg-1 dry weight (DW) and by enrichment with CO2 flue gases aims to produce Nannochloropsis for $1.00 kg-1 DW.

To lessen the costs associated with harvesting, Johnson (2009) provided “proof-of — concept” and cultured Chlorella sp. in the laboratory on attached solid polystyrene substrate on the bottom of a growth chamber. In dairy farm wastewater, Chlorella biomass reached production rates up to 3.2 g m-2d-1, comparable to suspended liquid cultures, and was harvested easily by scraping the solid surfaces. In addition to remediation of dairy manure water, an added advantage of this method was that the attached algal colonies served as inocula and eliminated the extra inoculation step (Johnson, 2009).

Although heterotrophic cultivation of algae could be cost-effective, only a few studies have been carried out. Growth of Chlorella vulgaris with the addition of the bacterium Azospirillum brasiliense in a heterotrophic regime, using glucose, yielded growth superior to that in cultures grown in autotrophic and mixotrophic regimes (Perez-Garcia et al., 2010). Chlorella protothecoides has been grown heterotrophi — cally using an organic carbon source in an enclosed environment of fermenters rang­ing from 5 to 11,000 L capacity (Li et al., 2007). The PBRs and fermenters have the advantages of reduced contamination and evaporation, but are more expensive than open ponds and raceways. They can be utilized in the production of large volumes of inocula while switching over from an autotrophic mode to heterotrophic mode of cultivation.

Under the temperate climatic conditions of British Columbia (Canada), calcu­lated base costs per liter of algal oil from raceways, closed PBRs, and fermenters correspond to $2.66, $7.32, and $1.54, respectively (Alabi, 2009), and fermenters are recommended. In temperate regions, the climate limits cultivation of algae in open raceways to the warmer seasons. Alabi (2009) summarized production costs for mass cultivation of autotrophic algae as $0.1 kg-1 to $32 kg-1 compared to the heterotrophic cultivation ($2.0 kg-1 to $12 kg-1). Algal-derived biofuel technology developed by Solix Biofuels costs about $33 per gallon, or about US$8 per liter (Kanellos, 2009). If biofuels are produced “dirt cheap” (Haag, 2007), production of fuel at an estimated cost of $50 or less per barrel (Huntley and Redalje, 2007) would be economically viable.