I.A.1.c. Collection and Screening Activities-1984 (cont)

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National Renewable Energy Laboratory

measured twice daily for 5 days, and the growth rates were calculated from the increase in culture

density during the exponential phase of growth. A refinement of this method was to measure the

growth rates in semi-continuous cultures, wherein the cultures were periodically diluted by half

with fresh medium; this method provided more reproducible results than the batch mode

experiments. Figure II.A.3 gives an example of the type of growth data generated by the use of

temperature-salinity gradient tables. The contour lines in the plot are interpolations that indicate

where a particular combination of temperature and salinity would result in a given growth rate.

Many such plots were generated for various strains, and are shown in the culture collection

catalogs and ASP annual reports.

Approximately 300 strains were collected from the 1984 trips to Utah and Colorado. Of these,

only 15 grew well at temperatures $ 301C and conductivities greater than 5 mmho$cm-1. Nine

were diatoms, including Amphora, Cymbella, Amphipleura, Chaetoceros, Nitzschia, Hantzschia,

and Diploneis.

Several chlorophytes (Chlorella, Scenedesmus, Ankistrodesmus, and

Chlorococcum) were also identified as promising strains, along with one chrysophyte

(Boekelovia).

Two strains isolated as a result of the 1984 collecting effort (Ankistrodesmus sp. and Boekelovia

sp.) were characterized in greater detail using the temperature-salinity matrix described earlier.

Boekelovia exhibited a wide range of temperature and salinity tolerance, and grew faster than

doubling$day-1 from 10° to 70 mmho$cm-1 conductivity and from 10 to 321C, exhibiting maximal

growth of 3.5 doublings$day-1 in Type II/25 medium. Reasonable growth rates were also

achieved in SERI Type I and ASW-Rila salts media (asmany as1.73 and 2.6 doublings@day-1,

respectively). Ankistrodesmus was also able to grow well in a wide range of salinities and

temperatures; maximal growth rates occurred in Type II/25 medium (3.0 doublings$day-1).

Boekelovia and Ankistrodesmus were also examined with regard to their lipid accumulation

potential. Two-liter cultures were grown in media that contained high (600 µM) and low (300

µM) urea concentrations at a light intensity of 200 µE$m-2$s-1. Half of each culture was harvested

2 days after the low-N culture entered stationary phase to determine the lipid content of N-

sufficient cells and cells that were just entering N-deficient growth. After 10 days of N-limited

growth, the remainder of the low-nitrogen culture was harvested. Lipids were extracted via a

modification of the method of Bligh and Dyer (1959) and lipid mass was determined

gravimetrically. The lipid content of Boekelovia was 27% of the organic mass in N-sufficient

cells, increasing to 42% and 59% after 2 days and 10 days of N-deficiency, respectively. There

was less effect of N starvation on the lipid content of Ankistrodesmus; the lipid content increased

only from 23% in N cells to 29% in cells that were N-deficient for 10 days.

In conclusion, research at SERI in 1984 led to the development of artificial media that mimicked

the saline groundwater typically found in the desert regions of the southwestern United States.

This allowed the strains isolated during collecting trips at various ionic concentrations to be

systematically screened and provided standardized media that could be used in different

laboratories performing ASP-sponsored research. Numerous strains were characterized with

respect to growth at several temperatures and salinities using these new media.

A Look Back at the Aquatic Species Program--Technical Review