The purpose of the research performed by Dr. Sriharan and coworkers was to study the effects of nutrient deprivation and temperature on growth and lipid production in microalgae with potential for liquid fuel production. All the reports generated by this laboratory during the subcontract describe a virtually identical set of experiments performed on several species of microalgae. The benefit of this approach is that the productivity data can be compared between the species. However, the flaws in experimental design and reporting were carried through all experiments and reports.

The basic design for these experiments was to grow the algal cells in batch cultures in media that contained either “sufficient” or “deficient” levels of N or Si, and to test for algal growth rate, productivity, and lipid production. Cultures were grown at 20°C and at 30°C to test for the effect of temperature on growth and lipid induction. Exponentially growing cells were inoculated into fresh media containing high or low levels of Si or N. Growth was monitored by measuring the optical density of the culture, and the growth rate was reported as the number of cell doublings per day. The cells were harvested and processed to determine lipid content (reported as a percentage of the AFDW), and fatty acid composition under the various growth conditions.

The organisms studied were all diatoms, except for the chlorophyte M. minutum (which the authors initially reported to be a diatom, but they corrected this error in a later report). The diatoms tested were Chaetoceros SS-14, C. muelleri var subsalsum, Navicula saprophila, obtained from SERI, Cyclotella DI-35, Cyclotella cryptica Reimann, Lewin, and Guillard, and Hantzchia DI-60, obtained from M. Tadros at Alabama A&M University. All the organisms tested grew more rapidly at 30°C (versus 20°C) and in nutrient-sufficient media. A decrease in the total AFDW was reported for all strains grown in nutrient-deficient media compared to nutrient-sufficient cultures, and this was accompanied by an increase in the percentage of the AFDW made up of lipids.

The most dramatic increases in the lipid content of the cultures were seen under N-deficient conditions in cells grown at 30°C. In C. cryptica, the total lipids, as a percentage of AFDW, increased from 15% to 44%. In Hantzschia, lipids increased from 29% to 53%, and in Navicula saprophila, lipids increased from 26% to 44%. In all cases, the increase in total lipids was due to increases in both the neutral lipid and polar lipid fractions. In several cases, the ratio of neutral

lipids to polar lipids increased significantly in the nutrient-stressed cells (i. e., in Hantzschia and C. muelleri grown in Si-deficient media, and in Navicula grown in N-deficient media). Dr. Sriharan also presented data comparing the fatty acid profiles of lipids from diatoms grown under nutrient-sufficient and nutrient-deficient conditions. Although the data was incomplete, it indicated that changes in the fatty acid composition (lipid quality) did occur in nutrient-stressed cells, suggesting that nutrient deprivation can affect the lipid biosynthetic pathways.

In all cases nutrient-deficiency resulted in a decreased rate of cell growth and a decrease in total cell productivity. Therefore, an increase in lipid as a percentage of cell mass may not be economically advantageous for liquid fuel production from mass-cultured algae if the conditions that induce lipid accumulation also result in a significant drop in total biomass, and thus in total lipid produced. Although this was not discussed by Dr. Sriharan, the total effect of nutrient limitation on lipid content of the algal cultures could be estimated by multiplying the total biomass (AFDW) produced by the percentage of the AFDW attributable to lipid under nutrient — sufficient or nutrient-deficient conditions. In general, these calculations demonstrated an increase in lipid content of the cultures induced by nutrient stress, in the range of a 20% to 30% increase in total lipid.

The results reported here clearly suggest that algal productivity is increased under nutrient — sufficient conditions and at elevated temperatures. However, it is difficult to determine the validity of the data presented regarding nutrient-deprivation as a lipid trigger. Growth curves were not presented for the organisms studied. Therefore, it cannot be determined if the low nutrient levels limited growth throughout the period of the experiment, or whether the nutrients became depleted and the lipid effects correlated with a decrease in cell division, as reported elsewhere. It is also not clear at what point in the cell cycle the cells were harvested for determination of AFDW and lipid content, and how to compare the data for nutrient-sufficient and nutrient-deficient cells. In several experiments, the authors reported that the cultures were only harvested when lipid droplets were seen in the cells, although this would seem to bias experiments designed to test nutrient effects on lipid production. All in all, it is difficult to determine whether the experiments were badly performed, or just poorly reported.

In summary, the data presented by Dr. Sriharan was difficult to interpret for these reasons; however, several general conclusions can be made. Diatoms seem to be promising candidates for neutral lipid production. Many species produce constitutively high levels of lipid, and the level of lipid as a percentage of biomass can be increased by growing the cells under nutrient-limited conditions (the data presented here suggests that N-limitation may be more effective than Si — limitation). In addition, Dr. Sriharan’s results suggest that nutrient-limitation may alter the lipid biosynthetic pathways in diatoms to increase lipid production and possibly affect lipid composition.