IV. A.1. Conclusions

From the early 1980s through the mid-1990s, there was a major effort by ASP researchers and contractors to identify or develop microalgal strains that demonstrated properties conducive to cost-effective biomass and lipid production. The characteristics deemed desirable in these strains include high productivity, high lipid content, competitiveness in outdoor culture, and tolerance to fluctuations in temperature and salinity. Although a number of strains were identified as possible candidates, no one strain was found to possess the optimal characteristics. As discussed elsewhere in this report, perhaps the most significant observation is that the conditions that promote high productivity and rapid growth (nutrient sufficiency) and the conditions that induce lipid accumulation (nutrient limitation) are mutually exclusive. Further research will be needed to overcome this barrier, probably in the area of genetic manipulation of algal strains to increase photosynthetic efficiency or to increase constitutive levels of lipid synthesis in algal strains.

The collection and screening efforts produced a number of significant findings. The SERI/NREL Microalgae Culture Collection was established as a valuable genetic resource and was the first microalgal collection that focused on organisms from brackish or saline environments. The organisms remaining in this collection (see Section II. A.3.) are being transferred to the University of Hawaii and should be available to interested researchers.

Although a number of algal strains were investigated for growth and lipid production properties, the best candidates were found in two classes, the Chlorophyceae (green algae) and the Bacilliarophyceae (diatoms). Organisms were identified in both classes that showed high productivity, ability to grow in large-scale culture, and lipid accumulation upon nutrient stress. However, in some ways the diatoms may turn out to be better candidate organisms for biofuels production. The highest lipid levels (40%-60% of the AFDW) were found in diatoms. Limiting the availability of Si, a major component of the diatom cell wall, can induce lipid accumulation in diatoms. In green algae, lipid accumulation is induced by N starvation. N is a component of many cellular molecules, and N limitation would induce a complex response, affecting photosynthesis, protein and nucleic acid synthesis, and other biochemical processes. In contrast, Si is not involved in most intracellular processes, so the response to Si limitation should be simpler to interpret and control. The disadvantage to diatom cultivation is the added cost of Si supplementation in the medium for optimal growth, although this could be minimized by the use of strains with lightly silicified walls. Some green algal strains, for example M. minutum, are advantageous for mass culture applications in that they can survive temperature fluctuations that are lethal for diatoms. We found however, that the properties that make these organisms very hardy, such as a very tough cell wall, also make their biochemical and molecular studies problematic. Despite these generalizations, the ideal organism(s) for a biofuels production facility will likely be different for each location, particularly for growth in outdoor ponds. The best approach will likely be to screen for highly productive, oleaginous strains at selected sites,

optimize growth conditions for large-scale culture, and optimize productivity and lipid production through genetic manipulation or biochemical manipulation of the timing of lipid accumulation in the selected strains. It is also likely that more than one strain will be used at a site, to maximize productivity at different times of the year.

Significant progress was made during the last 15 years in the understanding of lipid accumulation in the microalgae, although there is still much to be learned. Clearly microalgal cells can be induced to accumulate significant quantities of lipid when the medium is limited for an essential nutrient. However, the actual mechanism that triggers the accumulation is unclear. Lipid accumulation is correlated with the cessation of cell division. A simple explanation is that lipid synthesis continues in the non-dividing cells, but since no new membranes are being synthesized, the lipid is shunted into storage lipids. Alternatively, non-dividing cells are not utilizing cellular energy reserves as rapidly as dividing cells, so lipid accumulates as synthesis occurs more rapidly than utilization. Nutrient deprivation affects specific biochemical pathways, as lipid accumulation is accompanied by an increase in the proportion of storage lipids (TAGs) to polar membrane lipids, and Si deprivation in diatoms increases the expression of at least one gene involved in lipid synthesis, acetyl-CoA carboxylase. In general, nutrient deprivation induces lipid accumulation in cells and is accompanied by a decrease in total (and total lipid) productivity. However, studies of lipid accumulation suggest that an understanding of the kinetics of the process could be critical and could allow the identification of a stage where biomass productivity and lipid levels are optimal for maximal lipid accumulation.

Significant progress was also made in understanding the molecular biology of microalgae. Many of the green algae were found to contain DNA with unusually high GC ratios, and often with unusual modifications that would make these organisms more difficult as targets for genetic engineering. In contrast, the DNA content of diatoms is more typical of other eukaryotes. Work at NREL by ASP researchers resulted in the cloning and characterization of several genes involved in lipid and carbohydrate accumulation in diatoms, including the ACCase gene and a “fused” gene encoding the enzymes UDPglucose pyrophosphorylase and phosphoglucomutase. Isolation of these genes facilitated the development of a genetic transformation system for the diatoms. These genes were used in preliminary attempts to manipulate lipid production in these organisms. The successful development of the transformation system led to an increased understanding of the factors involved in introducing and expressing foreign genes in these organisms, and should facilitate the development of similar methods for other algal strains.

There is still much to be done in the area of microalgal strain development for lipid or biofuels production. A number of suggestions for possible research areas will be discussed in the following sections.