Previous to the research performed by researchers in the ASP at NREL, very little work had been done in the area of microalgal strain improvement, particularly with a goal of developing a commercial organism. Although much remains to be done, significant progress was made in the understanding of environmental and genetic factors that affect lipid accumulation in microalgae, and in the ability to manipulate these factors to produce strains with desired traits.

The evidence for a specific lipid trigger is not overwhelming. Interpreting exactly what is happening in the nutrient-deprived cells is difficult, particularly when cells are starved for N, as the lack of an important nutrient is likely to produce multiple and complex reactions in a cell. However, lipid accumulation in some algal species can be induced by nutrient limitation. Cell division slows or stops, and the cells begin to accumulate lipid as cytoplasmic droplets, formed primarily of neutral TAGs. The trigger hypothesis is supported by microscopic and flow cytometric evidence that showed that the lipid droplets do not form gradually within all cells in a population; rather, individual cells seem to sense the trigger and lipid accumulation occurs rapidly within the individual cells. However, lipid accumulation is always correlated with the cessation of cell division. Other factors that inhibit cell division, such as a pH shift, can also induce lipid accumulation in some strains. The evidence suggests that the rate of synthesis of all cell components, including lipids, proteins, and carbohydrates, is decreased in nutrient-stressed cells. However, the rate of lipid synthesis remains, at least for some strains of diatoms, higher

than the rate of protein or carbohydrate synthesis, resulting in a net accumulation of lipid in nutrient-starved cells. Another hypothesis is that cessation of cell division in nutrient-limited cells leads to decreased utilization of storage lipid while new synthesis of lipid continues, causing a net accumulation of lipid in the cells.

One of the most important findings from the studies on lipid accumulation in the microalgae is that, although nutrient stress causes lipid to increase in many strains as a percentage of the total biomass, this increase is generally accompanied by a decrease in total cell and lipid productivity. As discussed elsewhere in this report, economically viable production of algal lipids for fuel production will require optimization of productivity as well as a clear understanding of the kinetics of lipid accumulation, in order to harvest the cells at a time when lipid production is maximal.

In addition to the effect on total lipid production, nutrient deprivation seems to have other effects on the lipid biosynthetic pathways. Several laboratories reported that nutrient limitation also resulted in a change in the types of lipids seen in the algal cells, specifically, an increase in the ratio of neutral lipids (storage TAGs that are important in the production of biodiesel) as compared to the polar membrane lipids. It will be important to characterize this phenomenon further in any algal strain targeted for biomass production to maximize the desired lipid product.

The progress made by the ASP in the understanding of the biochemistry and molecular biology of lipid biosynthesis in algae, and the success in the area of algal genetic engineering are more clear cut. An enzyme that appears to play a key regulatory role in the synthesis of lipids in plants, and likely in algae, ACCase, was purified from the diatom C. cryptica. The gene that codes for this enzyme was then cloned; this was the first report of cloning a full-length ACCase sequence from any photosynthetic organism. Obtaining this gene was advantageous for two reasons:

1. The regulatory sequences from this gene were used to develop a genetic transformation system for diatoms, and

2. Having this gene (in combination with the transformation system) allowed researchers to test for the effects of overexpression of this enzyme on lipid accumulation.

The development of the transformation system for an oleaginous microalgal strain was a major goal of the ASP, and a significant effort was put into this project during the early 1990s. This was the first successful transformation of any non-green alga. The method was simple and reproducible, and should work for a variety of diatom strains, as long as the cells can form colonies on solid medium and are sensitive to one of the known selectable agents, such as G418 or kanamycin. Although little work is currently being done on the development of genetically engineered algal strains for commercial applications, the ability to transform these algae should have positive ramifications for the algal biotechnology community.

Preliminary experiments were also performed within the ASP to use this genetic transformation system to introduce genes into the algal cells, with the goal of manipulating lipid biosynthesis. Additional copies of the ACCase gene were introduced into cells of C. cryptica and N. saprophila. Although ACCase activity was increased in these cells, there was no detectable increase in lipid accumulation. The project was terminated before these experiments, and similar experiments designed to down-regulate genes involved in carbohydrate synthesis could be pursued further. This could be an interesting and possibly rewarding path for future research, if only to help in understanding the biochemical and molecular biological factors that affect lipid accumulation in these cells.

III. Outdoor Studies and Systems Analysis