The goal of the research performed by Dr. Schwartzbach and coworkers was to understand the biochemistry and physiology of lipid accumulation in microalgae, in particular the biochemical responses to N deficiency as a trigger for lipid accumulation. Lipid biosynthesis is dependent on the availability of fixed carbon and the activity of enzymes involved in lipid synthesis. These experiments were directed at understanding how these processes are affected by N limitation in the algal cells.

The first set of experiments analyzed lipid synthesis in the eustigmatophytes Nannochloropsis salina and Nanno Q, two oleaginous strains from the SERI Culture Collection. Similar results were obtained for the two strains. The basic protocol was to innoculate the algal cells into media containing either non-limiting levels or low levels of nitrogen (0.1 mM NaNO3), and to monitor cell growth, chlorophyll content, and the lipid levels per cell and per culture volume. In cultures containing low N, cell division ceased after 50-60 hours, and the cells entered stationary phase as the N was depleted. In contrast, cells grown with sufficient N continued to divide. In the N — replete cultures, the lipid content of the individual cells remained constant, and there was a steady increase in the amount of lipid per mL of culture as the cell number increased. In contrast, the N-deficient culture showed a significant increase in the level of lipid per cell. However, the lipid content of the culture per mL (or percentage of the AFDW composed of lipid) did not change. In N. salina, lipid made up 26%-32% of the AFDW, and in Nanno Q, lipid was 23%- 24% of the AFDW in cultures grown under N-replete and N-depleted conditions. These results indicate that N depletion causes the cells to stop dividing, while lipid synthesis continues. However, there is no net increase in lipid synthesis, and the trigger in these cells does not change the activity of enzymes involved in lipid biosynthesis. One caveat to these studies is that Dr. Schwartzbach measured only total lipid produced in the cells, including polar membrane lipids and nonpolar storage lipids; it is unclear from these studies and those described later whether N deficiency could differentially affect accumulation of the nonpolar lipids in these algae.

Another result from the studies on Nannochloropsis was that the level of chlorophyll in the cells declined rapidly in N depleted cells. Thus, N depletion would also presumably decrease photosynthetic efficiency and the availability of fixed carbon. The next set of experiments was designed to separate the effects of reduced photosynthetic efficiency from direct effects of N limitation on biosynthetic enzyme activities. To accomplish this, a series of experiments was performed using the eukaryotic green alga Euglena gracilis var. bacillaris Cori. Euglena can grow heterotrophically using ethanol as the sole carbon source. The growth of cells in the presence of externally supplied carbon (ethanol) should not be limited by decreased photosynthesis, so the rate of lipid synthesis would be solely limited by lipid biosynthetic capabilities.

Euglena is unique compared to most algae of interest to the ASP as potential producers of biodiesel. Euglena produces both lipid (primarily in form of the wax ester myristyl-miristate) and carbohydrate (the major product is paramylum, a P-1,3-glucan) as storage products. Using Euglena, a complicated series of experiments was conducted comparing the growth, lipid and carbohydrate content, and chlorophyll levels in algae under photosynthetic and heterotrophic growth conditions, as well as under aerobic and anaerobic conditions (Coleman et al. 1988b). Basically, cells were grown to N deficiency, then resuspended in fresh media containing either sufficient or limiting amounts of N. The new media also did, or did not, contain ethanol as a carbon source, and the cells were grown in the dark or in light.

As was seen with the Nannochloropsis strains, cell growth under N deficient conditions caused an increase in the levels of storage products (in this case, lipid plus carbohydrate) per cell. However, there was no net increase in total lipid/carbohydrate when measured as a percentage of
dry cell weight. This was true in cells grown autotrophically or heterotrophically. Nitrogen depletion caused the cells to stop dividing, but the storage products continued to accumulate in the cells at the same rate as in non-nitrogen limited cells. In addition, the proportion of carbohydrate and lipid was unchanged, thus there did not appear to be a N trigger effect, either directly or indirectly via carbon limitation, on the enzymes of the lipid or carbohydrate synthetic pathways. One caveat to this result was that in very old cultures, (i. e., 12 days after transfer of the cells to N-deficient media), the lipid as a percentage of the dry cell weight increased in all cultures. However, this was accompanied by a decrease in the total cell mass, and the lipids are apparently more stable than other cell components.

Growth of Euglena under N-deficient conditions resulted in loss of chlorophyll, as seen for Nannochloropsis. Dr. Schwartzbach also used two-dimensional gel electrophoresis to monitor changes in the levels of chloroplast and mitochondrial proteins under N-deficient conditions (Coleman et al., 1988a). Under photosynthetic growth conditions (high light), exposure of the cells to N-deficient conditions resulted in a decrease in the levels of 37 proteins identified as components of the chloroplast. Under low light conditions, there was little change in the population of chloroplast proteins. The degradation of the chloroplasts under low N conditions was presumably due to photooxidation of chlorophyll, accompanied by degradation of newly synthesized photosynthetic membrane proteins that could not assemble properly into the unstable chloroplast. Synthesis of chlorophyll requires N to form 5-aminolevulinic acid, a chlorophyll precursor. Although photooxidation of chlorophyll occurs constantly in the light, synthesis of new chlorophyll molecules also occurs to replace the degraded molecules. However, if N levels are depleted, new chlorophyll cannot be produced, and photosynthetic efficiency decreases. This result is important with regard to biodiesel production. It suggests that there would be limitations on the amount of lipid that could be produced in outdoor ponds using N limitation as a trigger for lipid accumulation even if carbon was not limiting (i. e., for cells grown in outdoor ponds).

One process that affected the biosynthetic pathways in Euglena and resulted in an increase in the total lipid in the cultures was cell growth under anaerobic conditions with ethanol as a carbon source (lipids increased from 5 -10% to 45% of the AFDW). Growth via anaerobiosis caused the activation of the oxygen-sensitive pyruvate dehydrogenase in the mitochondria. This led to increased levels of acetyl CoA in the mitochondria, which activates the mitochondrial fatty acid synthesis pathways. However, the increased flow of carbon to lipid synthetic pathways was accompanied by degradation of non-lipid components under anaerobic condition, including paramylum, the main storage carbohydrate, which resulted in a decrease in total cell mass. Dr. Schwartzbach estimated that if the anaerobic cells had increased in cell mass to the same extent as cells grown aerobically, the lipids would only compose 15% of the dry weight.

This observation that anaerobiosis could result in increased lipid yields by actually affecting the lipid biosynthetic pathway suggested that lipid synthesis could be increased by increasing the levels of the lipid precursors acetyl CoA and malonyl CoA. Little is known about lipid synthesis in algae, but data from other organisms suggested that pyruvate dehydrogenase and acetyl CoA carboxylase could function as regulatory enzymes in algal lipid synthesis. Understanding the biochemical factors that limit production of the lipid precursors could lead to biochemical or

genetic engineering strategies to increase the activity of these enzymes that could produce an organism with the ability to produce very high lipid levels. To this end, Dr. Schwartzbach initiated a project to isolate and characterize these enzymes from several algae, including Euglena, N. salina, Nanno Q, and Monoraphidium 2 (Smith and Schwartzbach 1988). They reported some very preliminary information on protein extraction techniques and assay techniques for these enzymes. This work was a precursor to a major effort at SERI/NREL in the late 1980s through the end of project in 1996 to identify key enzymes in the algal biosynthetic pathways and to increase lipid levels by manipulating these pathways through genetic engineering (see Sections II. B.2. and II. B.3.).

In summary, although Euglena is not typical of the oleaginous microalgae targeted as potential biodiesel producers by the ASP, the data from Dr. Schwartzbach’s laboratory point out the importanance of understanding the biochemical mechanisms by which algae accumulate lipids. For Euglena and the Nannochloropsis strains described here, N deprivation does not seem to function as an actual trigger to induce biosynthesis of lipid. Rather, it acts as a block to cell division. Lipid synthesis continues by normal pathways, and lipid levels increase per cell, with no net accumulation in the culture. This result confirms the conclusions of Cooksey and coworkers. In addition, N deficiency also affects other cell processes, such as photosynthetic efficiency, which could affect lipid accumulation as the availability of fixed carbon is decreased.

I Publications:

Coleman, L. W.; Rosen, B. H.; Schwartzbach, S. D. (1987a) “Environmental control of lipid accumulation in Nannochloropsis salina, Nanno Q and Euglena.” FY 1987 Aquatic Species Program Annual Report (Johnson, D. A.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, pp. 190-206.

Coleman, L. W.; Rosen, B. H.; Schwartzbach, S. D. (1987b) “Biochemistry of neutral lipid synthesis in microalgae.” FY 1986Aquatic Species Program Annual Report (Johnson, D. A., ed.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, p. 255.

Coleman, L. W.; Rosen, B. H.; Schwartzbach, S. D. (1988a) “Preferential loss of chloroplast proteins in nitrogen deficient Euglena.” Plant Cell Physiol 29:1007-101. (Note: a preprint of this article was also submitted as a SERI Report, 47pp.)

Coleman, L. W.; Rosen, B. H.; Schwartzbach, S. D. (1988b) “Environmental control of carbohydrate and lipid synthesis in Euglena” Plant Cell Physiol. 29:423-432.

Smith, C. W.; Schwartzbach, S. D. (1988) “Preliminary characterization of pyruvate dehydrogenase and acetyl-CoA synthetase.” Manuscript submitted as a report to SERI, 6 pp.