By 1987, SERI researchers and subcontractors had collected approximately 3,000 algal strains. Most of these strains had not been well characterized, especially with respect to lipid production capabilities. As a consequence, work commenced on the development of a simple screening procedure to estimate the lipid contents of cells to determine which strains had the best potential as biofuel production organisms. Ideally, the procedure should be simple and reproducible so that it could be used as a standard method in numerous laboratories. The researchers hoped that such a screening tool would allow the size of the strain collection to be reduced to a manageable number (~200) representing the most promising strains.

Development of a rapid screen for lipid content.

In an attempt to develop a reproducible, easy-to-use screening procedure to identify algal strains with high lipid contents, Dr. Keith Cooksey (an ASP subcontractor at Montana State University) suggested that investigators explore the possibility of using the lipophilic dye Nile Red (9- diethylamino-5H-benzo{a}phenoxazine-5-one) to stain cells. Nile Red was first isolated from Nile Blue by Greenspan et al. (1985), who showed that Nile Red will fluoresce in a nonpolar environment and could serve as a probe to detect nonpolar lipids in cells. Nile Red permeates all structures within a cell, but the characteristic yellow fluorescence (approximately 575 nm) only occurs when the dye is in a nonpolar environment, primarily neutral storage lipid droplets. Earlier work within the ASP by Dr. Steve Lien had shown the utility of Nile Blue in microscopically assessing the lipid content of algal cells (Lien 1981). The active ingredient in these Nile Blue preparations may in fact have been Nile Red. Parallel efforts to develop a Nile Red staining procedure were carried out by SERI researchers and ASP subcontractors, notably Drs. Cooksey and Sommerfeld.

Cooksey et al. (1987) used the diatom Amphora coffeiformis to optimize the Nile Red staining procedure. The dye was dissolved in acetone and used at a concentration of 1 mg/mL of cell suspension. In this species, the fluorescence of the dye in live stained cells was stable for only 2­7 minutes; fluorescence measurements had to be completed rapidly to ensure consistent results. The kinetics of fluorescence in stained cells varied in different species, presumably due to differences in the permeability of cell walls to the stain, and differences in how the lipid is stored in the cells, i. e., as large or small droplets. Fixing the stained cells with formaldehyde or ethanol preserved the Nile Red fluorescence for 2 hours, but cells that were chemically fixed before Nile Red staining did not exhibit the characteristic yellow fluorescence. When Nile Red fluorescence was measured in algal cultures over time, the fluorescence increased as the culture became N deficient. The fluorescence level was linearly correlated with an increase in the total lipid content, determined gravimetrically, in a growing culture of algal cells. Fractionation of the lipids by silicic acid column chromatography demonstrated that the increase in lipid was due primarily to an increase in neutral lipids rather than in the polar lipids or glycolipids, which are found primarily in cell membranes.

Additional development of the Nile Red screening procedure occurred at SERI and at Milt Sommerfeld’s laboratory at Arizona State University. The resultant protocol involved taking a fixed volume of a diluted algal culture (typically 4 mL), adding 0.04 mL of a Nile Red solution (0.1 mg/mL in acetone), and determining the fluorescence after 5 min using a fluorometer equipped with the appropriate excitation and emission filters.

Although the use of Nile Red allowed various microalgae to be rapidly screened for neutral lipid accumulation, interspecies comparisons may be subject to misinterpretation because of the species-specific staining differences described earlier. Nonetheless, before Nile Red was used, quantitating lipids from cells was very time consuming. It required the extraction of lipid from a large number of cells using organic solvents, evaporation of the solvent, and determination of the

amount of lipid by weighing the dried extract. Consequently, the use of Nile Red as a rapid screening procedure can still have substantial value.

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 mmhccm"1, both Type I and Type II media), high temperature (30°C), and high light intensity (average of 200 pE^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 subsequently used both in Milt Sommerfeld’s laboratory and at SERI to screen many microalgal isolates. Keith Cooksey’s laboratory also examined numerous strains using this procedure. Sommerfeld’s laboratory examined 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.

The lipid production potential of these strains was evaluated by the use of the fluorometric Nile Red assay. In Type I/55 medium, 49 strains had a higher apparent lipid content after 2 days of N-deficient growth, whereas 13 strains had the same or lower apparent lipid levels in response to N deficiency. In Type II/55 medium, 42 strains had higher lipid levels, and 7 strains had lower or unchanged lipid levels as a consequence of N deficiency. Of note was that the mean lipid level in cells grown in Type II/55 medium was nearly twice that of Type I/55-grown cells.

The strains exhibiting the highest Nile Red fluorescence levels are shown in Table II. A.3. All of these strains are diatoms, confirming the propensity of this group to accumulate lipids.

Table II. A.3. Strains from the Arizona State University collection having the highest Nile Red fluorescence.

These researchers also ranked strains according to the estimated lipid productivity of rapidly growing cells, based on the calculated growth rates and estimated lipid contents of exponential phase cells. The top strains resulting from this analysis are shown in T able II. A.4. However, the optimal strategy for maximizing lipid yield in actual mass culture facilities may require an “induction” step (i. e., manipulation of the culture environment, possibly involving nutrient deficiency). The ranking of strains would obviously be very different in that case.

Table II. A.4. Strains in the Arizona State University collection with the highest apparent lipid productivity during exponential growth, based on Nile Red staining.

SERI researchers also started to evaluate various strains by the rapid screening procedure. Initial work focused on 25 partially characterized strains. These strains were analyzed for growth and Nile Red fluorescence in exponentially growing cultures and in cultures grown under N-deficient conditions for 4 days. The results of the SERI and Sommerfeld laboratories cannot be compared directly, because Nile Red units are expressed differently and the time duration of N deficiency was not the same. The best strains of the 25 tested (based on the highest Nile Red fluorescence normalized to ash-free dry weight (AFDW) and rapid exponential growth) were determined to be CHAET9 (muelleri), NAVIC2 (Navicula saprophila), and NITZS12 (Nitzschia pusilla).

Twenty-eight strains of Chaetoceros were also examined using this screening protocol. The best strains indentified were CHAET21, CHAET22, CHAET23, and CHAET25 (all muelleri). All but the latter strain were isolated from various regions of the Great Salt Lake in Utah.

The departure of Dr. Bill Barclay and Dr. Jeff Johansen from the ASP, along with a greater emphasis on genetic improvement of strains, marked the end of the in-house collection and screening work. As a consequence, many of the 3,000 strains collected by ASP researchers during the course of this research effort were never analyzed via this rapid screening protocol. Nonetheless, enough strains had been analyzed at SERI and at the laboratories of various subcontractors to obtain a substantial number of promising strains. The next step was to determine their ability to grow in actual outdoor mass culture ponds. This work is described in Section III of this report.