The goal of the work performed under this subcontract was to collect and screen microalgal species from southern Florida. It emphasized collecting chromophytic algae (e. g., diatoms, chrysophytes, and prymnesiophytes), because this group of algae was known to often accumulate lipids. Collection trips were made in June and September 1985, and in February 1986 to the Florida Keys and the Everglades. Samples were taken from 123 sites, including various mangrove swamps, salt flats, canals, ditches, and shallow ponds. The basic physicochemical characteristics of the collection site waters were determined. The mean temperature was 29°- 30°C both for sites in the Florida Keys and the Everglades. The mean conductivity of the water from the Keys (35.6 mmho^cm-1) was somewhat higher than that of the Everglades (25.7 mmho^cm-1), whereas the pH values were similar (~8). To select for the fastest growing microalgal strains in the water samples, the original samples were enriched with nitrate, trace metals, and vitamins, and incubated under continuous light (880 pE^m-2^s-1, or 45% of full sunlight) at 30°C. The strains that became dominant in the cultures were isolated into unialgal cultures via micropipetting, serial dilution, and spreading onto agar plates. As a consequence of these experiments, 61 unialgal cultures were produced.

Preliminary evaluation of the growth of these strains in various media was performed in test tubes containing enriched seawater, SERI Type I/25, Type I/40, Type II/25, and Type II/40 media. The test tubes were incubated at 30°C under constant illumination at 300 pE^m-2^s-1. Growth rates were determined by measuring the OD750 every day for 5 days, and the final culture density was measured after 10 days. One hundred ten strains (including some strains already in

the Harbor Branch algal collection) were screened in this manner. In general, the strains that were newly isolated under the selection scheme outlined above grew more rapidly than the culture collection strains. Members of the Prymnesiophyceae, particularly coccolithophorids and ochromonads, tended to grow well in most media types, but the dinoflagellates isolated via these procedures did not grow well in the SERI standard media. Most species grew better in Type II medium than in Type I medium, although there were certainly exceptions to this. The highest growth rate (3.26 doublings^-1) was observed with a strain of Hymenomonas HB152 (HYMEN3) in Type II/25 medium. Seven strains had growth rates that exceeded 2 doublings^-1 in at least one media type; included in this group were Dunaliella HB37 (DUNAL2), Nannochloris HB44 (NANNO2), a yellow green unicell HB54 (UNKNO4), Chlorella HB82 (CHLOR7) and HB87, Pyramimonas HB133 (PYRAM2), and HYMEN3. Nine strains had growth rates of at least one doubling^-1 in all five media (including Chlorella HB84 (CHLOR8) and HB97 (CHLOR9), Nannochloris HB85 (NANO3), and all the strains mentioned earlier in this paragraph except for HYMEN3).

Several of the most promising strains were examined in more detail; they were grown in a matrix of five different salinities (8-60 mmho^cm-1) at five different temperatures (15°-35°C) by the use of a temperature-salinity gradient table, as described in previous sections. An artificial seawater medium (ASP-2) diluted with varying amounts of distilled water was used for these experiments. The cultures were exposed to constant illumination at 180 pE^m-2^s-1. Each of the four strains tested (Tetraselmis HB47 [TETRA4], PYRAM2, UNKNO4, and an olive green unicell HB154 [UNKNO5]) exhibited excellent growth over a wide range of conditions. All these strains had a growth rate greater than one doubling^-1 between 8 and 60 mmho^cm-1 and between 20° and 35°C. UNKNO4 had growth rates higher than 1.5 doublings^-1 between 15 and 60 mmho^cm-1 and between 20° and 35°C.

A visual assessment of the lipid contents of the most rapidly growing strains was conducted by staining the cells with Nile Red, the stained cells were examined using fluorescence microscopy. Based on this assessment (which was not carried out with nutrient-starved cells), TETRA5 and UNKNO4 had the highest estimated lipid contents. TETRA4, HYMEN2, PYRAM2, and UNKNO5 also appeared to accumulate substantial quantities of lipid.