creening for growth in high-conductivity media.

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National Renewable Energy Laboratory

Screening for growth in high-conductivity media.

The estimation of lipid content using a simple procedure such as the Nile Red assay is clearly an

important component of a rapid screening procedure for identifying promising strains, but an

equally important component is a means to identify strains that grow rapidly under the expected

culture conditions. Reports detailing the amount and types of saline groundwater available in the

southwestern United States, along with data concerning the high rates of evaporation in this

region, indicated that tolerance of algal strains to high conductivity (higher than 50 mmho$cm-1)

could be important. Therefore, an additional component of the secondary screening procedure

developed to reduce the number of strains being maintained by ASP researchers. Algae were

tested for the ability to grow at high conductivity (55 mmho$cm-1, both Type I and Type II

media), high temperature (301C), and high light intensity (average of 200 µE$m-2$s-1, 12 h

light:12 h dark cycle) in cultures that were continually agitated via aeration. To prevent osmotic

shock, strains were adapted to higher conductivities via a stepwise transfer into media with

increasing conductivity at 2-day intervals. Tubes were used that could be placed directly in a

spectrophotometer (i.e., 25 mm diameter, 50 mL volume), allowing the culture density to be

measured without removing a sample. The tubes also held enough medium to allow samples to

be taken for Nile Red lipid analysis (both for N-sufficient and N-deficient cells), and for ash-free

dry mass determinations. The tubes were placed in a rack and illuminated by fluorescent lamps

from below for the screening procedure. Optical density measurements were taken twice daily

for 4 days during exponential growth to determine growth rates. Samples were removed for Nile

Red fluorometric analysis during exponential growth and after 2 days (Arizona State University)

or 4 days (SERI) of N deficiency.

This newly developed rapid screening protocol was later used both in Milt Sommerfeld=s

laboratory and at SERI to screen many microalgal isolates. Keith Cooksey=s laboratory also

Sommerfeld=s laboratory examined

examined numberous strains using this procedure.

approximately 800 strains that had been collected over the previous 2 years of the subcontract.

Only 102 of these strains survived transfer into media having a conductivity of 55 mmho$cm-1.

Of these strains, 40 grew in both Type I/55 and Type II/55 media, 42 grew only in Type I/55

medium, and 19 grew only in Type II/55 medium. The 10 fastest-growing strains, along with their

preferred media, are shown in Table II.A.2.

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