The goal of this subcontract was to isolate and characterize strains of microalgae from the southeastern United States that have attributes desirable for a biodiesel production strain. During the first year of this work, field trips were made to several sites in Alabama to collect microalgal strains from a variety of habitats. Freshwater and brackish water strains were collected from rivers, lakes, estuaries, and ponds, and marine strains were collected from the waters surrounding Dauphin Island in the Gulf of Mexico. Collected samples were inoculated into various artificial media, including Bold’s Basal Medium, Chu no. 10, and “f/2” (Barclay et al. 1986). Artificial sea salts were used in place of seawater for the saltwater media. For initial strain selection, the cultures were incubated at 29-30°C with shaking at a light intensity of 100 to 125 qE^m-2^s-1 provided by cool white fluorescent bulbs with a 14 h:10 h light:dark cycle. The fastest growing strains were isolated via micropipetting or by spreading samples on agar plates. In these preliminary experiments, the marine strains exhibiting the fastest growth were Cyclotella DI-35 (CYCLO1), Hantzschia DI-160 (NITZS2), and Chlorococcum DI-34. The freshwater strains exhibiting the fastest growth rates were Chlorella MB-31, Scenedesmus TR-84, Ankistrodesmus TR-87, and Nitzschia TR-114.

CYCLO1, Nitzschia TR-114, and Scenedesmus TR-84 were selected for more detailed growth analyses under various combinations of temperature, salinity, and light intensity. A temperature gradient table was employed for these experiments that was similar in design to the tables used by Dr. William Thomas (discussed earlier) and SERI researchers for screening purposes. Growth of standing cultures was determined by measuring final cell densities after 12 days of incubation. CYCLO1 achieved maximum cell density at a temperature of 30°C, a light intensity of 100 qE^m-2^s-1, and a salinity of 15 ppt (parts per thousand). Growth was nearly as good at a light intensity of 200 qE^m-2^s-1 and a temperature of 35°C, and substantial growth occurred at a salinity of 32 ppt. Growth did not occur at 15° to 20°C. Nitzschia TR-114 achieved maximal cell density at 30°C, 15 ppt salinity, and 100 qE^m-2^s-1; growth was severely inhibited at 0 and 45 ppt salinity. Growth was similar at 100 and 200 qE^m-2^s-1 for this strain, although the higher light intensity seemed to increase the thermal tolerance of the cells. The freshwater strain Scenedesmus TR-84 grew best at 25°C, and grew increasingly slower as the salt concentration of the medium was increased.

The lipid contents of several strains isolated during the initial collecting trips were determined. For 14-day-old cultures that were reportedly N-limited (although no evidence is provided to support this), the lipid contents (as a percentage of the organic mass) were as follows: CYCLO1, 42.1%; Nitzschia TR-114, 28.1%; Chlorella MB-31, 28.6%-32.4%; Scenedesmus TR-84, 44.7%; Ankistrodesmus TR-87, 28.1%; and Hantzschia DI-160 (NITZS2), 66%.

Additional strains were collected the next year from intertidal waters near Biloxi, Mississippi and St. Joseph Bay, Florida. Preliminary screening experiments indicated that five strains (all of which were diatoms) had the best growth rates and lipid accumulation potential: Navicula acceptata (two strains, NAVIC6 and NAVIC8), N. saprophila (NAVIC7), Nitzschia dissipata (NITZS13), and Amphiprora hyalina (ENTOM3). These strains and CYCLO1 were grown semi­continuously in media with six different salinities at 25°, 30°, and 35°C. Cells were grown at light intensities of 80 qE^m-2^s-1 and 160 qE^m-2^s-1 (approximately 4% and 8% of full sunlight, respectively). The media were produced by adding various quantities of artificial sea salts to “f/2” medium; the resulting conductivities were <1, 10, 20, 35, 45, and 60 mmho^cm-1. (Note: seawater is typically 35-45 mmho*cm-1.) All strains exhibited more rapid growth under 160 qE^m-2^s-1 illumination than at 80 qE^m-2^s-1; and even higher growth rates might well have been obtained at light intensities greater than 160 qE^m-2^s-1.

ENTOM3 grew best (2.0 to 2.3 doublings^-1) at 30°C in media with conductivities of 20-60 mmho’cm’1. Growth was better with urea or nitrate as the N source rather than with ammonium. The lipid content of nutrient-sufficient cells was 22.1% of the organic mass, and increased to 37.1% and 30.2% under Si-deficient and N-deficient conditions, respectively.

CYCLO1 achieved the highest growth rates (2.8 to 3.0 doublings^-1) at 35°C between 10 and 35 mmho-cm-1. Cells grew best with nitrate as a N source, followed by ammonium and then urea. The highest lipid content was observed in N-deficient cells (42.1%), but was also elevated in Si — deficient cells (38.6%) relative to nutrient-sufficient cells (13.2%).

NAVIC8 grew most rapidly (3.8 doublings^"1) at 35°C and 45 mmho^cm"1. Nitrate and ammonium were more suitable N sources than urea. Lipid contents of 21.8%, 48.5%, and 32.4% were observed for cells grown under nutrient-sufficient, Si-deficient, and N-deficient conditions, respectively.

During the final year of this subcontract, additional promising strains were isolated. Included in this group was Navicula BB-324 (NAVIC9), which had a growth rate exceeding 2.5 doublings^-1 at 30°C in artificial seawater and SERI Types I/10, I/25, I/40, II/10, II/25, II/40, and II/55 media. Navicula SB-304 (NAVIC8) also exhibited excellent growth (1.5-3.0 doublings^-1) in each medium. These two strains had Si starvation-induced lipid contents of 42.5% and 47.2%, respectively. Other notable strains were Nitzschia SB-307 (NITZS13), which had a maximal growth rate of 2.5 doublings^-1 and a lipid content of 45%-47% under nutrient — stressed conditions. Amphiprora BB-333 (ENTOM3), Chaetoceros BB-330 (CHAET66), and Cylindrotheca AB-204 also grew rapidly (2.3-6.0 doublings^-1), with stress-induced lipid contents ranging from 16.5%-37.1%.

In conclusion, many promising strains were isolated as a result of this subcontract. The nutrient status of the cells again was played an important role in lipid accumulation. Furthermore, the nature of the N source included in the medium had a substantial impact on growth of the cultures. Several of these strains were further tested in outdoor mass culture, as described in Section III.