Scripps Institution of Oceanography Ralph A. Lewin 1985 — 1986 N/A

This subcontract focused on the collection and characterization of picopleustonic algae, which are defined as algae (including the prokaryotic cyanophytes) that are very small (1-5 pm) and that live on the surface of the water. In February 1985, water samples were taken from various sites in the Caribbean Sea, including sites near the U. S. Virgin Islands (St. John, St. Thomas, and St. Croix), Tortola, Puerto Rico, Curasao, Panama, and the Florida Keys. 130 samples (250 mL each) were collected and filtered through a 3-8-pm filter to remove larger cells. Smaller cells were collected on a 0.45 pm nitrocellulose filter, which was rolled up and placed in the original sampling water that had passed through the filter. These samples were placed under natural lighting at 20°C to 25°C until transported to the laboratory. The filters were then transferred into a tube of sterile enriched seawater (containing additional N and other nutrients) and incubated at 25°C under continuous illumination from a fluorescent lamp at 30 pE^m-2^s-1. In an attempt to stimulate lipid accumulation via nutrient deficiency, a portion of each culture was transferred after 4 weeks of growth to a fresh tube of unenriched seawater and then allowed to grow under the same conditions. After 4 more weeks, a film of cells was often observed floating on the surface of the cultures. Small samples of these cells were transferred to fresh enriched seawater. After incubation for an additional 2 weeks, the cells in these cultures were microscopically examined, and cultures that were dominated by diatoms, cyanophytes, and flagellates were

discarded, leaving approximately 60 cultures of small (1-5-pm) green cells. Unialgal cultures were established from these cells by isolating colonies on agar plates. Of these purified cultures, there were 14 isolates of Stichococcus, 21 isolates ofNannochloris, four strains of Chlorella, and several representatives of other genera. Stichococcus, Nannochloris, and Chlorella are all chlorophytes. Because cyanophytes typically do not accumulate lipids, they were eliminated from further study in this subcontract. The researchers anticipated that isolating strains in this manner would enrich for lipid-accumulating microalgae.

The isolated strains were tested for the ability to grow in freshwater; all the Stichococcus and Chlorella strains grew well in freshwater, suggesting perhaps a brackish water origin for these strains. Only six of the 21 Nannochloris strains could grow on non-marine media.

To quantitatively determine lipid content in the isolated strains, 1-L cultures were grown for 3 weeks in enriched seawater under continuous illumination at 30 pE^m-2^s-1. The cultures were bubbled with 0.5% CO2 in air. Cells were harvested, frozen and lyophilized, and then extracted three times with a chloroform/methanol mixture (2:1 v/v). After the solvents evaporated, the lipid mass was determined gravimetrically and normalized to the cellular AFDW. The 13 Stichococcus strains had lipid contents ranging from 9% to 59% of the AFDW, with an average of 33%. The lipid contents of the 21 Nannochloris strains ranged from 6% to 63%, with an average of 31%. Data were not presented for the lipid contents of the four Chlorella strains, although three strains of the eustigmatophyte Nannochloropsis that were isolated from Qingdao, China were examined; for this genus, the lipid content ranged from 31% to 68%, with an average of 46%. These reported lipid contents may be slight overestimates, in that there was apparently no attempt to remove somewhat polar materials that may have also been extracted via the use of an aqueous washing step.

Some preliminary experiments were also conducted during the course of this subcontract regarding the growth of the eustigmatophyte Nannochloropsis (strain Nanno-Q, one of the Qingdao strains). This strain is euryhaline, and is able to grow in seawater as well as brackish water with one-tenth the salinity of seawater. The cells grew to a higher final yield with nitrate as the N source than with ammonium, and the lipid content rose substantially when the N source was initially supplied at levels below 200 pM (as determined by the percentage of cells that were floating due to elevated lipid levels). A number of Nannochloropsis strains that had been obtained primarily from the Culture Collection of Marine Phytoplankton at Bigelow Laboratory (West Boothbay Harbor, Maine) were analyzed with respect to maximum cell yields after 4 weeks of growth at different temperatures. Most of the strains had temperature optima at or below 25°C, although one strain that had been collected near Long Island, New York had a temperature optimum of 33°C.

This subcontract examined a group of microalgae that had not received much attention in the ASP until that point. The small size of picopleustonic algae could hinder harvesting efficiency in a mass culture facility, which would have a negative impact on the economics of biodiesel production. However, if the cells could be made to consistently float due to high lipid levels, this

property might facilitate harvesting. Outdoor testing of the most promising strains would help to evaluate this group of microalgae.

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