Subcontractor: Principal Investigator:

Montana State University Keith E. Cooksey

Period of Performance: 1/86 — 10/87

Subcontract Numbers: XK-5-05073-1; XK-6-05073-1

The goal of this research was to understand the biochemistry of lipid accumulation in microalgae, in particular, to provide information on the biochemical triggers that induce lipid synthesis. The Nile Red fluorescence technique developed by Dr. Cooksey’s laboratory and at SERI (described above in Sections ILA. Lf. and III. B.1.e.) was used to study lipid accumulation in microalgae in response to N or Si depletion. Nile Red fluorescence was used to monitor the lipid levels in batch cultures of Chlorella over time. As the N became depleted, lipids accumulated in the cultures, predominately as triglycerides. The triglyceride levels began to increase before N was totally depleted from the medium. Microscopic examination showed that individual cells within the population began to accumulate lipid at different times, similar to results obtained by Dr. Solomon in Isochrysis. Dr. Cooksey concluded that lipid accumulation begins as the cells enter stationary phase and cell division ceases; the timing of this event would be different for individual cells within a population.

Dr. Cooksey’s laboratory next performed a complex series of experiments designed to correlate media factors, (i. e., nitrate concentrations, pH, and carbon availability), with lipid accumulation in CHLOR-1. Cell growth and lipid accumulation were monitored in batch cultures, with cells grown in unbuffered Bold’s medium, or media buffered at pH 7, 9, or 10. In unbuffered Bold’s medium, the initial pH was 7, and increased to pH 8 by day 6, and up to pH 9.5 by day 9. The cells grew in all conditions, with the best growth at pH 9. Under all growth conditions, the level of nitrate in the media decreased, but never fell below 25% of the initial levels.

Accumulation of neutral lipids was monitored by Nile Red fluorescence. There was no lipid accumulation in cultures maintained at or below pH 9. However, in buffered medium with a pH> 10, or in unbuffered medium that experienced an increase in pH during the growth period, the cultures generally showed a significant increase in lipid levels that was accompanied by a decrease in cellular growth rates.

Nutrient limitation, generally nitrate or silica, can trigger lipid accumulation in microalgae. Nutrient deprivation can cause a decrease in cell division, which presumably results in “targeting” of excess fixed carbon into storage lipids. The data obtained by Dr. Cooksey’s laboratory suggested that a shift in pH, which has been correlated with decreased rates of cell division, could also trigger lipid accumulation. These data suggested that nutrient limitation might not directly affect biochemical pathways to enhance lipid synthesis; rather, lipid accumulation may be an indirect consequence of inhibition of a stage in the cell cycle. In other photosynthetic systems studied, the data indicated that cells synthesize triglycerides in the light and utilize these lipids as energy stores in the dark and during cell division. If division were

blocked, the rate of neutral lipid utilization would be slower than the rate of synthesis, so triglycerides would accumulate in the cells. To help test this hypothesis, Dr. Cooksey used light microscopy to examine cells grown in media with different pH ranges. Cultures grown at pH 7-9 consisted almost entirely of small, single cells. However, at pH 10 and higher, a large proportion of the cells was in the form of autosporangial complexes, i. e., their nucleii had divided, but the autospores had not separated. The specific effect of increased pH on cell division is not clear, although some evidence suggests that increased pH can lead to increased flexibility of the autospore wall, preventing individual cells from breaking free. Alternatively, increased pH could affect precipitation of media components, indirectly affecting the cell cycle.

Although the data presented here suggest that nutrient deprivation or increased pH may affect lipid levels simply as a consequence of decreased cell division, additional research by Dr. Cooksey’s laboratory suggested that treatments that increase lipid accumulation may also affect the biochemistry of lipid biosynthesis. Analysis of the lipid classes present in the cells at the end of the 10-day growth period showed accumulation of triglycerides in cells at high pH, with a decrease in glycolipids and polar lipids. The nonpolar storage lipids predominantly contain 16- and 18-carbon saturated or monounsaturated fatty acids (16:0 and 18:1), which are considered “precursor” fatty acids in lipid biosynthesis. The polar lipids and glycolipids usually contain a higher proportion of polyunsaturated fatty acids. However, analysis of the fatty acid composition of the storage lipids showed that at higher pH, more of these precursor lipids were seen in the polar lipids and glycolipids. This suggests a switch in the lipid synthesis patterns that results in less desaturation of the fatty acids esterified to the polar lipids.

In summary, the finding that an increase in pH can also lead to lipid accumulation in cells before N is depleted suggested a method to uncouple lipid accumulation from nutrient deprivation, and provided another method to study the biochemistry of lipid accumulation in microalgae. The data from Dr. Cooksey’s laboratory supported the premise that lipid triggers such as nutrient deprivation or pH increase affect lipid accumulation in microalgae by similar mechanisms, i. e., inhibition of cell division, leading to decreased utilization of storage lipid while new synthesis of lipid continues. However, he also proposed that different stresses may affect different stages of the cell cycle. As there is evidence that the different lipid classes (neutral lipids versus polar lipids) may be synthesized at different times during the cell cycle, this could affect the quality and the quantity of the lipids synthesized. For example, pH stress appears to block release of autospores (after DNA replication); N deprivation could have multiple effects on the photosynthetic machinery or on a number of biochemical pathways in the cell that could directly or indirectly affect lipid synthesis. R. Thomas, a graduate student working with Dr. Cooksey, found that treating the cells with monofluoroacetate (MFA) also decreased cell growth and caused neutral lipid accumulation. MFA inhibits the TCA cycle, presumably preventing TCA oxidation of fatty acids and thus increasing the pool of acetyl CoA for synthesis of new fatty acids. (Thomas suggested that MFA could be used as a trigger for lipid accumulation in algal ponds, with the caveat that MFA is toxic to all living systems).

Dr. Cooksey concluded that to understand the biochemistry of neutral lipid accumulation in microalgae, it would be necessary to understand cellular cycles of lipid synthesis and utilization

that are coupled to cell growth and division. It will also be important to consider not only factors that affect synthesis of storage lipid, but also to understand the metabolic shifts that result in production of membrane lipids or storage lipids.

I Publications:

Cooksey, K. E. (1987) “Collection and screening of microalgae for lipid production.” Final Subcontract Report, Solar Energy Research Institute, Golden, Colorado, May 1987, 42 pp.

Cooksey, K. E.; Guckert, J. B.; Thomas, R. (1989) “Triglyceride accumulation and the cell cycle in microalgae.” Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, pp. 139-158.

Cooksey, K. E.; Guckert, J. B.; Williams, S. A.; Collis, P. R.(1987) Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red, J. of Microbiol. Methods 6:333-345.

Cooksey, K. E.; Williams, S. A.; Collis, P. R. (1987) “Nile Red, a fluorophore useful in assessing the relative lipid content of single cells,” In The Metabolism, Structure and Function of Plant Lipids, (Stumpf, P. K; Williams, S. A.; Callis, R. P., eds.), Plenum Press, N. Y., pp. 645-647.

Guckert, J. B.; Cooksey, K. E.; Jackson, L. L. (1987) “Lipid solvent systems are not equivalent for analysis of lipid classes in the microeukaryotic green alga, Chlorella.” Unpublished manuscript.

Guckert, J. B.; Cooksey, K. E. (1990) “Triglyceride accumulation and fatty aid profile changes in Chlorella (Chlorophyta) during high pH-induced cell cycle inhibition.” J. Phycol. 26:72-79.

Thomas, R. M. (1990) “Triglyceride accumulation and the cell cycle in Chlorella.” Masters Thesis, Montana State University, July 1990.