Of the species examined, P. tricornutum and T. sueica had the highest overall productivities. These species also had the highest lipid productivities, which were 4.34 and 4.47 g lipid^m-2^d-1, respectively. For both species, the maximal productivities were obtained in batch cultures, as opposed to semi-continuous or continuous cultures. Although the lipid contents of cells were often higher in response to N deficiency, the lipid productivities of all species tested were invariably lower under N deficiency because of an overall reduction in the culture growth rates. For the species tested under continuous or semi-continuous growth conditions, lipid productivities were reduced from 14% to 45% of the values measured for N-sufficient cultures.

The results also pointed to the importance of identifying strains that are not photoinhibited at light intensities that would occur in outdoor ponds. Finally, this work highlighted the fact that some microalgae accumulate carbohydrates during nutrient-deficient growth; such strains are clearly not acceptable for use as a feedstock for lipid-based fuel production.

(Also see references listed in the following section.)

II. A.2.c. Selection of High-Yielding Microalgae from Desert Saline Environments

University of California, San Diego William H. Thomas 1983 — 1986 XK-2-02170-0-01

The work carried out under this subcontract represented one of the first efforts to collect microalgae from inland saline habitats and to screen those strains for rapid growth rates and lipid content. Collecting trips were made to eastern California and western Nevada, and initial culturing efforts were conducted at the Sierra Nevada Aquatic Research Laboratory near Mammoth Lakes, California, and the Desert Studies Center (Zzyzx Springs), which is near Baker, California. Various saline waters and soils were sampled during these collecting trips. The collection sites included Pyramid Lake, Black Lake, Owens Lake, Walker Lake, Saline Valley, Zzyzx Springs, Armagosa River, Sperry River, Harper Lake, and Salt Creek. The water samples were enriched with N, P, Si, and trace metals, then incubated under natural conditions. The algae that grew up were isolated by the use of micropipettes. Soil samples were placed in “Zzyzx medium” before algal isolation (see Thomas et al. [1986] for media compositions).

Diatoms, green algae, and cyanobacteria were the dominant types of algae isolated using these procedures. Of the 100 strains isolated, 42 were grown under standardized conditions in various

artificial media that were designed to mimic the water from which the strains were originally obtained. The pH of these various media formulations was typically high because of the presence of high levels of carbonate and bicarbonate, and the total dissolved solids ranged from approximately 1.5 g^L-1 to over 260 g^L-1. The growth of the cultures was visually scored, and nine of the fastest growing strains were further analyzed with respect to growth under scaled-up conditions.

For larger scale cultures, 6 L of medium that was enriched with N (nitrate, urea, or ammonium), phosphate, trace metals, and vitamins were placed in 9-L serum bottles, and the cultures were illuminated with fluorescent bulbs at a light intensity of 18% full sunlight. To enhance growth, the cultures were bubbled with 1% CO2 in air, and more nutrients were added as the cell density of the cultures increased. The strains tested in this manner included Nitzschia, Ankistrodesmus, Nannochloris, Oocystis (two strains), Chlorella (three strains), and Selenastrum. The estimated productivities ranged from 8.8 g dry weighHm-2M-1 for for Nitzschia S-16 (NITZS1[5]) grown in the presence of urea to 45.8 g dry weighHm-2M-1 for Oocystis pusilla 32-1, which was also grown with urea as the N source. (These productivity values were considered overestimates in that there was incidental side lighting of the flasks under the incubation conditions.) Also the productivity values did not always correlate with final biomass yield values, indicating that growth saturation was reached at different culture densities for the various strains. The maximum biomass yield was obtained with O. pusilla 32-1 (2.29 g dry weighHL-1). The results of these experiments indicated that certain strains had a clear preference for either urea or nitrate as the N source. Because urea is significantly less expensive than nitrate, these results have economic implications with respect to algal mass culture. However, the results regarding a preference for a particular N source were not always reproducible.

Additional experiments were carried out to assess the combined effect of temperature and salinity on the growth of several of the isolates. A thermal gradient table was used for these experiments in which the incubation temperature at six stations on the table varied between 11 °C and 35°C. Salinities of the media were varied in five increments along the other axis (as much as twice that of the natural waters), leading to a total of 30 different combinations of temperature and salinity. Growth was determined via optical density measurements, and contour lines were drawn upon a matrix chart of the various temperature/salinity combinations. This approach was later used by other subcontractors and SERI in-house researchers to determine the optimal growth characteristics of numerous promising algal strains. Results from this analysis were reported for eight different strains. In general, the strains grew better at higher salinities, indicating their halophilic nature, and had temperature optima for growth between 20°C and 30°C. Of the strains tested, the Mono Lake isolate NITZS1 had the highest temperature optimum (between 30 and 36°C).

The effect of light intensity on the growth of Ankistrodesmus falcatus 91-1 (ANKIS1, from Pyramid Lake) and O. pusilla 32-1 (from Walker Lake) was determined in 0.83-L cultures that were placed at varying distances from a tungsten lamp source. Neutral density filters were

placed between the light source and the cultures. This arrangement provided between 30% and 70% of full sunlight. For ANKIS1, maximum productivity (21.9 g dry weight^m-2^d-1) was attained when the cells were subjected to 50% full sunlight. At 30% full sunlight, the productivity fell off to 14.7 g dry weight^m-2^d-1 (because of light limitation), and at 70% full sunlight, the productivity was reduced to 19.0 g dry weight^m-2^d-1 (most likely because of photoinhibition). For O. pusilla, a maximum productivity of 25.8 g dry weight^m-2^d-1 was also attained at 50% full sunlight, whereas productivity at 30% and 70% full sunlight was 18.7 and 23.2 g dry weight^m-2^d-1, respectively. These productivity values are believed to be more accurate than those reported in the preceding section because light was able to enter the culture vessels only from one side. The relative high densities of these cultures (>1 g dry weights-1) permitted the cells to tolerate higher light intensities than would be possible in less dense cultures, because of the self-shading of the cells.

Experiments were also conducted to determine the effects of varying the culture vessel width (i. e., culture depth) on overall productivities. ANKIS1 was grown in containers that were 5, 10, and 15 cm wide. The containers were illuminated with a tungsten lamp at 50% full sunlight, and were bubbled vigorously with 1% CO2 in air. The results of these experiments indicated that growth rate, when expressed as g dry weight^L-1^d-1, was highest in the 5-cm thick culture and lowest in the 15-cm thick culture. However, when productivities were expressed as g dry weight^m-2^d-1, which takes into account the actual surface area that is illuminated, the thicker cultures were more productive. For example, the volumetric productivities over 10 days were 0.72, 0.35 and 0.31 g dry weight^L-1^d-1) for the 5-, 10-, and 15-cm thick cultures, respectively, whereas the corresponding areal productivities for these cultures were 41.1, 40.2, and 52.7 g dry weight^m-2^d-1. Because the economic constraints regarding an actual algal biodiesel production facility dictate the use of open pond systems, the areal productivity values are the more important consideration, although less water (and consequently less water handling) is required when dealing with more dense cultures. The productivities reported for these experiments may be overestimates of what these strains could achieve in outdoor mass culture because of the optimized mixing and aeration regime.

In the final year of this subcontract, additional collecting trips were taken to gather more microalgal strains. These strains, along with some that had been collected during earlier trips, were screened more rigorously than before. In this revised selection process, the strains were subjected to higher light intensities and higher temperatures, and the abilities of the strains to grow in “SERI standard media[6]“ were investigated. This selection procedure resulted in the isolation of 41 additional strains. Initial screening of strains involved incubating the isolates at 25 °C and 30°C under 40% full sunlight (provided by a 2000-W tungsten-halide lamp) in the SERI standard medium that most closely resembled the water from which they were originally isolated. Twelve of the strains that grew best under these conditions were then tested under the same temperature and light conditions in an early version of standard SERI media (Type I and Type II at low, medium, and high salinities; see media compositions in Thomas et al. [1985]).

The results indicated that most of the strains had a definite preference for a particular medium type and level of salinity. The results also indicated some inconsistencies in the growth rates of cells grown in the two experiments. For example, Chlorella BL-6 (CHLOR2) grew very well in the preliminary experiments in Type II/low salinity medium (2.48 doublings^-1), but grew much more poorly when grown in all five SERI media (including Type II/low salinity) in the second set of experiments. Conversely, Chlamydomonas HL-9 grew much more quickly in the second set of experiments than in the first set. The reasons for these discrepancies are unclear, as the culture conditions were essentially the same, and underscore the need to perform replicate experiments. Several marine microalgae were also tested for the ability to grow in SERI standard media. Phaeodactylum tricornutum, Chaetoceros gracilis, and Platymonas all grew well (>1.25 doublings^-1) in at least one SERI medium. Isochrysis T-ISO was unable to grow in any SERI medium, however.

The combined effects of temperature and salinity on the growth rates of eight of these newly collected strains were determined by the use of a temperature-salinity gradient table. In general, the strains grew best in the range of salinity that was similar to that of the water from which they were originally isolated. The optimal temperature for growth was generally in the 25°C to 35°C range, although one Chlorella strain from Salt Creek grew well at 40°C. Based on the results of these experiments, two strains were selected for analysis of growth characteristics in larger scale (12 L) cultures at 50%-70% full sunlight. CHLOR2 achieved a productivity of 55.5 g dry weight^m-2^d-1 under these conditions, and Nannochloris MO-2A had a productivity of 31.9 g dry weight^m-2^d-1.

This subcontract represented one of the first efforts in the ASP to collect and screen microalgal strains to identify suitable biofuel production strains. As a consequence, many of the screening and characterization protocols were still being developed; therefore, there is a substantial lack of uniformity in the testing of the various strains isolated. Nonetheless, a number of promising strains were isolated during the course of this research, and several methods were developed that helped establish standard screening protocols used by other ASP researchers.

Publications:

Thomas, W. H.; Gaines, S. R. (1982) “Algae from the arid southwestern United States: an annotated bibliography.” Report for Subcontract XK-2-0270-01. Solar Energy Research Institute, Golden, Colorado, October 1982.

Thomas, W. H.; Seibert, D. L.R.; Alden, M.; Eldridge, P.; Neori, A.; Gaines, S. (1983b) “Selection of high-yielding microalgae from desert saline environments.” Aquatic Species Program Review: Proceedings of the March 1983 Principal Investigators ’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 97-122.

Thomas, W. H. (1983b) “Microalgae from desert saline waters as potential biomass producers.”

Progress in Solar Energy 6:143-145.

Thomas, W. H.; Seibert, D. L.R.; Alden, M.; Eldridge, P. (1984c) “Cultural requirements, yields, and light utilization efficiencies of some desert saline microalgae.” Aquatic Species Program Review: Proceedings of the April 1984 Principal Investigators ’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, pp. 7-63.

Thomas, W. H.; Tornabene, T. G.; Weissman, J. (1984d) “Screening for lipid yielding microalgae: Activities for 1983.” Final Subcontract Report. Solar Energy Research Institute, Golden, Colorado, SERI/STR-231 -2207.

Thomas, W. H.; Seibert, D. L.R.; Alden, M.; Eldridge, P. (1985) “Selection of desert saline microalgae for high yields at elevated temperatures and light intensities and in SERI Standard artificial media.” Aquatic Species Program Review: Proceedings of the March 1985 Principal Investigators ’Meeting, Solar Energy Research Institute, Golden, Colorado SERI/CP-231-2700, pp. 5-27.

Thomas, W. H.; Seibert, D. L.R.; Alden, M.; Eldridge, P. (1986) “Cultural requirements, yields and light utilization efficiencies of some desert saline microalgae.” Nova Hedwigia 83:60-69.