Subcontractor: Principal Investigator: Period of Performance: Subcontract Number:

Arizona State University Milton Sommerfeld 1985 — 1987 N/A

The objectives of this subcontract were to collect microalgal strains from a variety of locations in the desert regions of the Southwestern United States and to screen them for their ability to grow under conditions in a commercial microalgal biodiesel facility. Studies were also conducted to optimize a fluorometric procedure for estimating cellular lipid content, and to use this method to screen some of the strains.

Collecting trips took place between April 1985 and June 1986. Water samples containing microalgae were collected from 125 sites in Arizona, California, Nevada, New Mexico, Texas, and Utah. Some samples were taken from saline surface waters in the regions of Arizona and New Mexico that were deemed suitable for microalgal mass culture, based in part on the availability of large quantities of saline groundwater (Lansford et al. 1987). Researchers believed that strains from these areas would be well adapted to the indigenous waters available for mass culture. These areas included the Palo Verde Irrigation District in Arizona, and the Pecos River Basin, the Crow Flats area, and the Tularosa Basin in New Mexico. The temperature, pH, specific conductance, and water depth were recorded at each collection site. Most of the waters sampled had a specific conductance exceeding 2 mmho^cm-1. T emperatures ranged from 18°C to 45°C (mean = 26.9°C), pH ranged from 6.1 to 10.2 (mean = 8.0), and specific conductance ranged from 0.45 mmho^cm-1 to 474 mmho^cm-1 (mean = 22.7 mmho^cm-1).

Planktonic algae were the primary type of alga collected, although neustonic and benthic forms were also collected when algal growth in such habitats was clearly visible. From these samples, more than 1,700 strains of microalgae were obtained. From these strains, approximately 700 unialgal cultures were established. Initial strain isolations were performed by streaking out samples onto 1.5% agar plates containing seawater, sterilized collection site water, or SERI Type I or Type II medium having a conductivity similar to that of the collection site water. In some cases, an enrichment step was performed before streaking out the cells, wherein the samples were placed in tubes on a rotary agitation wheel in liquid media at a light intensity of 1500 pE^m-2^s-1 (~75% of full sunlight) using a 12h:12h light:dark cycle.

Of the 700 unialgal cultures, 120 were identified taxonomically; 24 genera were represented in this group, including 60 chlorophytes, 40 diatoms, and 20 cyanophytes. The most common genera were Dunaliella, Chlorococcum, Chlorosarcina, Amphora, Nitzschia, Navicula, Oscillatoria, and Chroococcus. Initial screening typically involved visual assessment of growth at 25°C at 200 pE^m-2^s-1 in tubes containing SERI Type I/40 and Type II/40 media. Strains that grew the most rapidly were subjected to further characterization, including analysis of the effects

of temperature, salinity, light intensity, and N source on growth rates. Cultures were grown in several different media (SERI Types I and II media at various conductivities, and artificial seawater) at 30°C and 500 pE^m-2^s-1 using a 12h:12h light:dark cycle on a rotary screening apparatus. Thirty-one diatoms were tested under these conditions, and 11 exhibited growth rates exceeding one doubling^-1. The highest growth rate observed for a diatom under these conditions was 1.96 doublings^-1 for Amphora ASU0308. Of the 50 chlorophyte species tested, 17 strains exhibited growth rates exceeding one doubling^-1; the highest growth rate (2.58 doublings^-1) was observed for a strain of Dunaliella (ASU0038). Of the strains that were tested for growth in all seven standard media, 80% of the cultures grew in the low salinity SERI Type I and Type II media and more than 50% of the strains grew in seawater. The highest growth rates were typically observed in SERI Type I/10 and Type II/10 media and seawater, although the mean growth rates of all strains combined at the highest salinities (70 mmho^cm-1) were 60% to 80% of the mean growth rates obtained at the lower salinities. Most of the strains were isolated from waters with specific conductances below 40 mmho^cm-1, which may explain the lower growth rates in the media having higher salinities. A few strains, however, grew quite well in the higher salinity media. Amphora ASU0032 (AMPHO27), Synechococcus ASU0071 (CHLOC5), also referred to by the subcontractor as Chroococcus, and Navicula ASU0267 were the only strains that had a growth rate that exceeded one doubling^-1 in Type II/70 medium. Certain strains had high growth rates in both Type I/70 and Type II/70 media; included in this group were the strains mentioned earlier along with Synechococcus ASU0075 (CHROC2) and Dunaliella ASU0038. Some strains were clearly euryhaline (i. e., able to tolerate a wide range of salinities), and could grow in all media tested (e. g., CHLOC5 had a growth rate that exceeded one doubling^-1 in each of the seven media tested). Other strains were stenohaline, and grew much better at one particular salinity. Certain strains showed no real preference for SERI Type I versus SERI Type II medium, despite the very different ionic composition of these media types. Other strains exhibited a clear preference for one media type over the other (e. g., Dunaliella ASU0038 grew much better in SERI Type I medium or seawater than in SERI Type II medium). Twenty-eight of the strains tested had growth rates exceeding one doubling^-1 in at least one media type, three strains had growth rates that exceeded two doublings^-1, and one strain (either Eremosphaera or Chlorococcum ASU0132 [CHLOC6] or ASU0048 [CHLOC4]) had a growth rate higher than three doublings^-1.

The proximate chemical compositions of 11 isolates were also determined in this study. Total lipid was determined by the Bligh-Dyer procedure (Bligh and Dyer 1959). Protein was determined by the heated biuret-Folin assay, and total carbohydrate was estimated by the phenol- sulfuric acid method (see Sommerfeld et al. [1987b] for details). Of the newly isolated strains tested, Franceia ASU0146 (FRANC1) had the highest lipid content under normal growth conditions (26.5% of the AFDW). The strains were not evaluated under nutrient-deficient conditions, which often increases the lipid content of microalgae. Because analyzing the lipid content of all the strains that had been isolated by this type of procedure would be very difficult, the strains were examined by the use of lipophilic dyes. The dyes Nile Blue A, Sudan Black B, Oil Red O, and Nile Red were used in conjunction with fluorescence microscopy to check for oil droplets within the cells. Inconsistent results were obtained when using the first three stains, but Nile Red appeared to give more reliable results. The stained cells were visually scored for the

presence of fluorescing lipid droplets. Additional work was carried out to develop a Nile Red staining procedure that could (in theory) provide a quantitative measure of lipid content by the use of a fluorometer. This latter work was discussed in Section II. A.1. along with the results of similar efforts carried out by SERI researchers and other subcontractors.

This subcontract was somewhat unusual in that many of the strains were collected from the actual areas in which the commercial microalgal biodiesel facilities could be. As a consequence, many of the strains that were isolated had characteristics that could make them good candidates for production strains. Additional results from this subcontract are presented in Section II. A.1.