Oak Ridge National Laboratory, Oak Ridge, Tennessee Jean A. Solomon 10/84 — 11/86 N/A

The goal of this project was to gain further understanding of the physiology of lipid accumulation in microalgae by examination of the ultrastructure of cells containing high levels of storage lipids. The questions that Dr. Solomon addressed were:

1. Where does the lipid accumulate within the cells; and

2. What other ultrastructual changes are seen in microalgae induced to accumulate lipid?

Three oleaginous microalgal strains were used in this study, Ankistrodesmus fulcatus (SERI strain ANKIS1; class Chlorophyceae), Isochrysis aff. glabana (ISOCH1, class Prymnesiophyceae), and Nannochloropsis salina (NANNO1, class Eustigmatophyceae). Ultrastructural changes were monitored by transmission electron microscopy (TEM). In this technique, cells are chemically fixed and embedded in a plastic resin. The resin is then cut into thin sections (70-100nm), stained with heavy metals, and viewed in an electron microscope. The first step was to develop adequate fixation and embedding techniques for the algal species to be studied. This is often problematic for microalgae, presumably due to the chemical and physical properties of the algal cell wall, which can act as barriers to penetration of the fixatives or resin. Dr. Solomon tested five fixation protocols (see Solomon 1985, p.74, Table 1), all variations of standard methods of fixation using glutaraldehyde and osmium tetroxide, dehydration with an organic solvent, embedding of the cells in an acrylic resin, and poststaining of the sections with uranyl acetate and lead citrate. Initially, Dr. Solomon reported that the best fixation of Ankistrodesmis and Isochrysis was achieved by exposing the cells briefly to glutaraldehyde and osmium simultaneously, followed by dehydration in acetone and embedding in Spurrs resin. However, a later report stated that Ankistrodesmis was better preserved by exposing the cells sequentially to glutaraldehyde and then osmium (in cacodylate buffer supplemented with sucrose as an osmoticum). Also, Araldite/Embed12 resin was used, as it appeared to provide better penetration into the cells. For Isochrysis, the initial protocol was also modified by adding sucrose. Fixation of Nannochloropsis was poor with any method used; the scaley cell wall of this organism seemed to provide a significant barrier to adequate penetration of fixatives and resins.

Nitrogen deprivation was used to trigger the production of lipids in the cells. The cells were grown in N-replete medium, then collected by centrifugation and resuspended in growth medium without added N. Samples were fixed immediately and at regular intervals during the following 13 days, and thin sections were cut and examined for ultrastructual changes by TEM.

As expected, N deprivation resulted in the accumulation of lipid within the cells of all three microalgal species. The lipid appeared primarily as droplets within the cytoplasm, not within the chloroplast or other cellular organelles. The lipid droplets often appeared adjacent to a mitochondrion. In Ankistrodesmus, N-deficiency also produced an increased number of starch granules within the chloroplasts, and resulted in the formation of unusual membrane structures consisting of packed, concentric layers of double membranes within the cytoplasm. Whether these unusual structures were the site of excess lipid accumulation, or were structural artifacts of the fixation process, was unclear.

It is difficult to conclude much more about ultrastructural changes that might have been induced in these cells following N deprivation. The sample size was very small. One hundred-nm thick sections may represent less than 1/100th of the volume of a microalgal cell. In addition, only a few cells within a population can be examined easily by this technique. Finally, there is a high likelihood that the chemical fixation methods used in the study can create artifacts that are not related to actual cell structure. However, these studies supported the observation that significant levels of storage lipids can accumulate in the cytoplasm of microalgal cells exposed to N deficiency. Dr. Solomon’s microscopic observations in Ankistrodesmus also suggested the presence of a discrete lipid trigger mechanism within each cell, as lipid did not appear to accumulate gradually within all cells of a population after N deprivation. Instead, individual cells appeared to accumulate large amounts of lipid during a 1-2 day period. This result was supported by the flow cytometric data also performed in Dr. Solomon’s laboratory, which is described below.

I Publications:

Solomon, J. A.; Hand, R. E.; Mann, R. C. (1986b) “Ultrastructural and Flow Cytometric Analyses of Lipid Accumulation in Microalgae: A Subcontract Report.” Solar Energy Research Institute, Golden, Colorado, SERI/STR-231-3089.

Figure II. B.2. Electron micrographs of nitrogen-sufficient (top) and nitrogen-deficient (bottom) cells of Nannochlorposis salina.

Note the accumulation of large lipid droplets (L) in the cytoplasm in the nitrogen-deficient cells. The lipid often appeared adjacent to a mitochondrion (M). N: nucleus. C: chloroplast. Scale bars = 0.5 pm. The numbers in the lower left corner of each figure are from the original publication (Solomon et al. 1986b).