Análisis, interpretación de los resultados

The screening and characterization protocols used by SERI researchers were refined for the 1984 collecting season. Included in these refinements was the development of a modified “rotary screening apparatus”, a standard type of motorized culture mixing wheel for 16x150-mm culture tubes. The rotating wheel was constructed of Plexiglas to allow better light exposure (see Figure II.A.1). The wheel was typically illuminated with a high-intensity tungsten stage lamp, and

could be placed inside a box behind a CuSO4-water heat filter for temperature control. The Plexiglas wheel allowed all the cultures to receive equal illumination. Another technological advance used a temperature-salinity gradient table to characterize the thermal and salinity preferences and tolerances of the isolates.

Development of artificial saline media.

One of the most significant contributions made by SERI researchers during 1984 was the development of media that mimicked the saline water in shallow aquifers in the southwestern United States. This was an important undertaking because it allowed algal strains to be screened for growth in the types of water that would likely be available in an outdoor mass culture facility. To identify the major water types available in the southwestern United States, state and federal reports that described the chemical characteristics of water from 85 saline wells in New Mexico were studied. The data were statistically analyzed to identify the relationships between the various ionic constituents. (Data from wells deeper than 83 m was not used in this analysis, because the cost of pumping water from those depths was prohibitive.) R-mode factor analysis indicated that two factors were largely responsible for the differences between the waters examined (Barclay et al. 1988). The first factor, monovalent ion concentration, was responsible for 40% of the variance; the second factor, divalent ion concentration, for 30%. A plot of these factors against each other clearly delineated two primary water types, referred to as “Type I” and “Type II”. Type I waters were characterized by a low monovalent-to-divalent ion ratio (average value = 0.4), whereas Type II waters had a higher level of monovalent ions (monovalent-to-

divalent ion ratio of 9.4). The major ions present in Type I water were Na+, Cl-, Mg2+, and Ca2+.

The major ions of Type II water were Na+_{, Cl}-_{, SO}

42-, and HCO3-. Type II water is consequently

termed a “sodium bicarbonate class” of water. Approximately three-fourths of the saline well waters were of the Type II variety, and one-fourth could be characterized as Type I.

The survey indicated that both types of water exhibited a range of conductivities; the researchers believed that the higher-conductivity waters resulted from evaporation of the lower conductivity waters. In addition, they recognized that the conductivity of the water in an outdoor production pond would increase with time because of the high rates of evaporation in the southwestern

United States (as high as 1 cm•day-1_{). Therefore, artificial media that covered a wide range of}

conductivities had to be developed. To this end, an experiment was conducted in which media that contained the salts typically present in low-conductivity Type I and Type II waters were allowed to evaporate with stirring at 35ºC. Samples were removed at various times and filtered. The ions still dissolved in the waters were quantified using an inductively coupled plasma spectrometer and a high-performance liquid chromatograph. In this manner, media formulations

were derived at SERI that covered a range of conductivities (from 10 to 70 mmho•cm-1_{) for both}

media types. The media most commonly used were designated SERI Type I/10, Type I/25, Type I/55, Type I/70, Type II/10, Type II/25, Type II/55, and Type II/70, in which the number following the slash indicates the specific conductivity of the medium. The compositions of these media are given in Figure II.A.2.

In order to assess whether these media formulations accurately reflected the types of water in desert region surface waters, samples of the water at numerous algal collection sites in the southwestern United States were chemically analyzed. The relative compositions of the anionic and cationic constituents were then plotted on separate trilinear plots, which allowed a graphical representation of the various water samples relative to SERI Type I and Type II media (Figure

II.A.3). This analysis indicated that Type I water has higher proportions of Mg2+ _{and Ca}2+ _than

most surface waters examined, whereas Type II water was fairly representative of the sampled waters with respect to these cations. On the other hand, natural surface waters often had an anion composition similar to both SERI Type I and Type II media. The researchers concluded that these artificial media would serve well as standardized media for testing newly acquired strains, thereby allowing all ASP researchers (both in-house personnel and subcontractors) to screen strains for growth potential in waters similar to those that would be available for commercial production.

Collection activities.

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 µM urea, 30 µM

PO4, 36 µM Na2SiO3, 3 µM 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 25ºC or 30ºC) and illuminated at ~400 µE•m-2•s-1. Over a 5-day period, the illumination

provided by the stage lamp was gradually increased to 1,000 µE•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 25ºC at 150-200

µE•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

µE•m-2•s-1. Thirty combinations of temperature (10º to 35ºC) 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 27ºC. 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 ≥30ºC 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 10º to 32ºC, 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 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-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.

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.” FY 1986 Aquatic 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 1986 Aquatic Species Program Annual Report, Solar

Energy Research Institute, Golden, Colorado, SERI/CP-231-3071; pp. 53-66. 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. 18:159-

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

Figure II.A.3. Trilinear plots showing the ionic constitutents 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 of Monoraphidium 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