SERI researchers Lien and Roessler (1986) tried a somewhat different approach to understand the processes affecting lipid accumulation (Lien and Roessler 1986). A recently published technical evaluation (Hill et al. 1984) identified two major requirements for economic feasibility of biodiesel production:

1. Photosynthetic efficiency (which can simply be thought of as the percentage of incident radiation that is converted into biomass) needs to be 18%, and

2. Algal biomass needs to consist of 60% lipid.

Because very high lipid production is usually correlated with stress conditions (nutrient deprivation) that result in decreased photosynthetic efficiency and decreased growth, the two conditions of high lipid and high productivity seemed to be mutually exclusive. To overcome this technical hurdle, Lien and Roessler initiated a study to help understand the effects of nitrogen deprivation and lipid accumulation on photosynthetic efficiency.

Three strains of oleaginous algae were used in this study: Chlorella CHLSO1, Ankistrodesmus sp., and a newly isolated chrysophyte strain Chryso/F-1. The cells were grown in batch culture and monitored for nitrate concentration, light levels in the culture, chlorophyll concentration, and yield of cell mass and lipid (including total, neutral, and polar lipids). Maximum energy efficiency occurred as the culture approached N depletion. At this point, the culture showed a maximum density of photosynthetic pigments (before chlorophyll degradation and after N depletion), but the light energy reaching the cells was decreased due to the higher culture density. Thus, photosynthetic efficiency (biomass produced per light energy input) was maximized and the individual cells suffered less photooxidative damage due to lower light exposure. After the N in the culture was depleted, cell mass continued to increase for a time, eventually leveling off. All cultures experienced a two — to three-fold increase in total lipid, primarily as non-polar lipid. The photosynthetic efficiency decreased over the duration of the batch culture. However, in the early stages after the N was depleted, the cultures showed a decrease in energy efficiency with respect to total cell mass (AFDW) and with respect to the non lipid cell components, while photosynthetic efficiency remained constant or increased slightly with respect to lipid accumulation. In addition, N deprivation caused an increase in the efficiency of neutral, storage lipid production and suppressed the efficiency of polar structural lipid production.

These studies provided interesting preliminary data on the energetics of cell mass and lipid accumulation in algae. Follow-up experiments were proposed, including investigations of the relationship between initial N concentration and photosynthetic efficiency and lipid production after N depletion, and studying the effects of N resupply after depletion to attempt to extend the period of lipid production. These experiments were not continued; however, the results described earlier suggest that understanding the timing or kinetics of lipid accumlation in microalgae will be essential to maximize lipid production in a mass culture facility. If N starvation is used to trigger lipid accumulation, the data suggest that maximal photosynthetic

efficiency with respect to lipid production (and probably the best time for harvesting lipid- producing cells), occurs just after the N is depleted from the cultures.

Another set of experiments directed at optimizating photosynthetic efficiency in algal ponds was performed by SERI researcher Dr. Ken Terry. Previous studies had indicated that algal cells grown under high-intensity flashing light can use that light energy more efficiently than cells grown under the same intensity under constant illumination. The evidence suggests that an algal cell can integrate absorbed light energy such that the photosynthetic efficiency achieved under intermittent light conditions is similar to that attained under constant light of the same average intensity. This flashing light, or photomodulation, effect can be mimicked in vertically-mixed algal ponds, as cells circulate to the surface and back down to the lower levels in the pond where they receive minimal light. Thus, the photosynthetic efficiency of algal cells grown in ponds may be increased in high light by using mixing strategies that optimize this photomodulation effect.

In order to better understand the effects of intermittent light on photosynthetic efficiency of microalgal cultures, Dr. Terry set up a system to measure photosynthetic rates and oxygen evolution in laboratory cultures of Chlorellapyrenoidosa and Phaeodactylum tricorutum under flashing light conditions. Intermittent light conditions were simulated by placing sectored disks in front of a light source, and using this to illuminate exponentially growing cultures that had been placed in an oxygen electrode chamber. Photosynthesis was then measured under varying light/dark ratios (generated by changing the configuration of the disk) and light intensities. The data generated were used to calculate the percent “integration” of the incident light by the algal cultures. More rapid flashing led to greater integration, although lower flash frequencies produced higher levels of integration as the percentage of time the cells spent in the light decreased. Although these data were preliminary, they supported the proposal that photosynthetic efficiency in microalgal ponds could be enhanced by optimized vertical mixing strategies. However, increased photosynthetic efficiency might be compromised by increased losses to respiration as the cells spend increased time away from the surface, and the energy costs to achieve optimal mixing could be prohibitive.

Although Dr. Terry proposed follow-up studies using modulated light regimes that more closely mimic those seen in algal ponds, little further research on understanding photosynthetic efficiency in algal cultures was performed at SERI. Instead, the emphasis of the in-house research shifted to understanding the biochemistry and molecular biology of lipid accumulation.