Collecting trips made by SERI researchers in 1984 focused on shallow saline habitats, including ephemeral ponds, playas, and springs in the arid regions of Colorado and Utah. After collection, the water and sediment samples were kept under cool, dark conditions for 1 to 3 days until they could be further treated in the laboratory. The pH, temperature, conductivity, redox potential, and alkalinity of the collection site waters were determined, and water samples were taken for subsequent ion analysis. In the laboratory, the samples were enriched with 300 pM urea, 30 pM PO4, 36 pM Na2SiO3, 3 pM NaFeEDTA, trace metals (5 mL/L PII stock, see Figure II. A.2), and vitamins. The enrichment tubes were then placed in the rotary screening apparatus (maintained at 25°C or 30°C) and illuminated at ~400 pE^m-2^s-1. Over a 5-day period, the illumination provided by the stage lamp was gradually increased to 1,000 pE^m-2^s-1. The predominant strains present in the tubes were isolated as unialgal cultures by agar plating or by serial dilution in liquid media.

The isolated strains were then tested for their ability to grow in incubators at 25°C at 150-200 pE^m-2^s-1 in the standard media types described above. and in artificial seawater (termed “Rila Salts ASW,” using Rila Marine Mix, an artificial sea salt mixture produced by Rila Products, Teaneck, NJ. The strains that grew well in at least one of these media were further characterized with respect to growth on a temperature-salinity gradient table at a light intensity of 200 pE^m-2^s-1. Thirty combinations of temperature (10° to 35°C) and salinity (10 to 70 mmho’cm’1) were included in this analysis, representing the ranges that might be expected in actual outdoor production systems. Once again, the cultures were enriched with nutrients to maximize growth rates. The cultures used to inoculate the test cultures were preconditioned by growth in the media at 17° and 27°C. The optical density at 750 nm (OD750) of the cultures was 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 semicontinuous 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 indicating 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 >30°C and conductivities greater than 5 mmho^cm-1. Nine were diatoms, including the genera 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 one doubling^day-1 from 10 to 70 mmho^cm-1 conductivity and from 10° to 32°C, 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 (as many as 1.73 and 2.6 doublings^day-1, respectively). Ankistrodesmus was also able to grow well in a wide range of salinities and temperatures, with maximal growth rates occurring 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 pM) and low (300 pM) urea concentrations at a light intensity of 200 pE^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-N 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 only increased from 23% in N-sufficient 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.

I Publications:

Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, R.; Lemke, P. (1986) “Microalgae Culture Collection 1986-1987.” Solar Energy Research Institute, Golden, Colorado, SERI/SP — 232-3079, 147 pp.

Barclay, B.; Nagle, N.; Terry, K. (1987) “Screening microalgae for biomass production potential: Protocol modification and evaluation.” FY1986Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-3071; pp. 23-40.

Barclay, B.; Nagle, N.; Terry, K.; Roessler, P. (1985) “Collecting and screening microalgae from shallow, inland saline habitats.” Aquatic Species Program Review: Proceedings of the March 1985 Principal Investigators ’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700; pp. 52-68.

Barclay, W. R.; Nagle, N. J.; Terry, K. L.; Ellingson, S. B.; Sommerfeld, M. R. (1988) “Characterization of saline groundwater resource quality for aquatic biomass production: A statistically-based approach.” Wat. Res. 22:373-379.

Sommerfeld, M. R.; Ellingson, S. B. (1987) “Collection of high energy yielding strains of saline microalgae from southwestern states.” FY 1986Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-3071; pp. 53-66.

I Additional References:

Bligh, E. G.; Dyer, D. J. (1959) “A rapid method for total lipid extraction and purification.” Can. J. Biochem. Physiol. 37:911-917.

Siver, P. (1983) “A new thermal gradient device for culturing algae.” British J. Phycol. 18:159­163.

Figure II. A.1. Rotary screening apparatus used for microalgal screening.

SERI Type I

Artificial Inland Saline Water

Salt	10	23	40	55	70
CaCl2………………………….		3,932	5,618	7,610	2,430
MgCl2-6H20……………..		11,844	22,789	35,305	42,230
N^2^0^………………………		2,923	3,310	3,705	3,620
KCI…………………………….		407	662	960	1,126
NaHC03……………………..		l£g	162	162	162
NaCl…………………………..		3,S45	9,132	13,023	16,039
		0	0	0	0

Conductivity (mmho cm’*)

SERI Type П

Artificial Inland Saline Water

Conductivity (mmho cm’1)

Salt

CaCl,

NajSOj,.

KCt….

iidnwj.

NajCOj <

Suggested enrichments (mL/L) are:

Nitrogen source* (0.6M N). KH2PO^(0.6M) a

Bj2<l mg L’1) і……………………………………………………

Thiamine-HCI (i mg L’*)…………………………………..

Biotin (2 mg L’1)………………………………………………..

•Nitrogen source indicated for individual species, ammonium as NH^Cl, nitrate as KNO3. 230-500 mg L’* Na2Si03.9H20 should be added when cultivating diatoms in this medium.

РП trace element stock (lor 1 U:

Na2EDTA…………………………………………………. 6.0 g

FeCtj-CHjO………………………………………………. 0.29 s

H3B03……………………………………………………… 6Л4&

MnCyWjO……………………………………………….. Q. S6 I

ZnCl2 …………………………….. …………………… 0.0({

CoCl^tHjO……………………………………………….. 0.026*

Adjust trace element stock solution to pH 7.8-S. O with NaOH.

Figure II. A.2. Formulations for SERI Type I and Type II artificial inland saline waters.

Recipes for the preparation of Type I and Type II media at five different salinities, expressed as conductivity of the final solution. Formulas for these media were developed by statistical analysis of saline groundwater data for the state of New Mexico. For each salt, necessary additions in mg/L are listed. (Source: Barclay et al. 1986).

Figure II. A.3. Trilinear plots showing the ionic constituents of various water samples relative to SERI Type I and SERI Type II artificial saline media. (Source: Sommerfeld and Ellingson 1987.)

Figure II. A.4. Growth contour plots. Examples of growth contour plots generated from data obtained by the use of a temperature-gradient table. The contour lines represent interpolated values indicating where a particular combination of temperature and salinity would result in a given growth rate. The data shown, given as doublings»day) represent the exponential growth ofMonoraphidium sp. (S/MONOR-2) in semicontinuous culture. Each point represents the mean of at least five separate daily growth rate determinations. (Source: Barclay et al. 1987).

A: Type I inland saline water B: Type II inland saline water C: Seawater