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A note added to a chapter of the 1986 Annual Report (Lien and Roessler 1986) described preliminary data on the use of Si deficiency to trigger lipid accumulation in diatoms. Silicon is major component of diatom cell walls. Similar to the lipid trigger effect produced by N- deficiency, Si depletion also results in a decrease in cell growth and often is accompanied by an accumulation of lipid within the cells. However, Si (unlike N) is not a component of other cellular macromolecules (enzymes, membranes) or cell structures such as the photosynthetic

apparatus. Therefore, any changes in cellular biochemistry and lipid accumulation induced by Si deficiency might be more easily interpreted than changes induced by N starvation. This work initiated a series of experiments by Paul Roessler during the late 1980s and early 1990s on the biochemistry and molecular biology of lipid accumulation in Si-deficient diatoms.

The first set of experiments compared the effects of Si deficiency on lipid accumulation and cell

physiology in several species of diatoms, including C. cryptica T13L, Thalassiosira pseudonana,

and Cylindrotheca fusiformis. Exponentially growing cultures were transferred to media that contained either excess Si or limited levels of Si so that the media became Si deficient while the

cells were still growing exponentially. Cell growth, chlorophyll a content, AFDW, lipid, and

photosynthetic capacity were monitored under both conditions. In all three species, cell division decreased as soon as the Si was depleted in the media. However the species responded

differently with respect to other physiological parameters. In C. cryptica, chlorophyll a synthesis

was almost completely inhibited after 12 hrs in Si-depleted media; C. fusiformis showed little

change in chlorophyll a synthesis after 72 hrs. T. pseudonana exhibited an intermediate effect,

with some decrease in chlorophyll a synthesis noted after 36 hours without Si. The effect on

photosynthetic capacity, measured as O2 evolution, also varied between the three species. In C.

fusiformis and C. cryptica, photosynthetic capacity decreased 33% and 58%, respectively, after

12 hours; T. pseudonana showed a steady decline in photosynthetic capability following Si-

depletion. (However, photosynthetic capacity decreased in Si-replete cultures as well during the 72 hours time course of the experiment, presumably due to the increased ratio of antenna chlorophyll molecules versus reaction center molecules in the self-shaded, dense cultures). The three species were also analyzed for accumulation of total biomass and lipid (Figure II.B.3.). In C. fusiformis, biomass accumulation (measured as AFDW) for the duration of the experiment was similar in cultures with or without sufficient Si, although lipids made up a higher percentage

of the AFDW in the Si-deficient cultures (26% versus 21% in Si-replete cells). In T.

pseudonana, synthesis of cell mass and lipid was not affected until 36 hours after Si depletion. At this point, biomass and lipid accumulation rates decreased; however, there was little difference in the percentage of total lipid in the cells with or without Si at the end of the 72 hours

experimental period. The situation with C. cryptica was very different. Twelve hours following

Si depletion, there was a 38% decrease in the growth rate of these cells compared to the Si- replete culture. However, lipid synthesis continued at the same rate in the Si-deficient cells as in the Si-replete cells, resulting in a significant increase in the lipid content of the Si-starved cells.

Interestingly, after these initial changes, the Si deficient cultures of C. cryptica showed little gain

in total AFDW or lipid during the remaining 72 hours of the experiment.

In order to determine if Si deprivation affected the composition of the lipids produced, the lipids were extracted and analyzed for the percentage of polar versus neutral lipids present. In all three species, the Si-deficient cultures showed a significant increase in the level of neutral lipids,

primarily TAGs. For example, the percentage of neutral lipids in Si-deficient cultures of C.

cryptica was 64%, compared to 32% in Si-replete cultures. In C. fusiformis, the percentage of neutral lipids increased from 17%-20% to 57% in Si-deprived cultures.

Based on these studies, C. cryptica was identified as the best candidate for further studies on the biochemistry of lipid accumulation. To determine the effects of Si deficiency on the synthesis of the cell components, the levels of protein, carbohydrate, and lipid were examined at various times after Si was depleted in the cultures. During the first 12 hours, protein and carbohydrate synthesis decreased. Lipid accumulation continued at a rate similar to that of the Si-replete cultures. This resulted in an increase in lipid content of the Si deficient cells from 19% to 27%. This observation was confirmed in subsequent studies that followed the incorporation of newly

assimilated carbon (as H14_CO

3-) into the various cell components. Si depletion resulted in a net

decrease in the rate of photosynthesis and carbon assimilation, but the individual cell fractions

were affected differently. For example, the rate of 14_{C accumulation into lipids decreased by}

48% in the first 4 hours of Si-deprivation; the uptake of 14_{C into chrysolaminarin, the major}

carbohydrate storage product in diatoms, decreased 84%. Therefore, the increase in lipid content of Si-deficient cells was not due to an increase in the rate of lipid synthesis, but to a relative decrease in the rate of synthesis of protein and carbohydrate.

Pulse-chase experiments were performed to test whether Si deficiency also caused the conversion of non-lipid cellular components into lipids. In these experiments, Si-replete cells were labeled

with H14_CO

3- for 1 hour, then transferred into Si-deficient media without labeled bicarbonate.

The amount of labeled carbon in the lipid fraction was determined at various times following transfer to Si-free media. This experiment showed that carbon was slowly redistributed from the nonlipid components of the cells into lipid under Si-deficient conditions, but not under Si-replete conditions. Therefore, the accumulation of lipids in diatoms in response to Si-deficiency is apparently due to two factors:

1. An increase in the proportion (but not the net amount) of newly assimilated carbon that is incorporated into lipids, resulting from a disproportionate decrease in the rate of lipid synthesis versus carbohydrate synthesis, and

2. A slow conversion of nonlipid cell material into lipids.

Fractionation of the lipids produced demonstrated that Si deprivation resulted in an increase in the proportion of total lipid as neutral lipids, primarily TAGs, from 43% to 63% after only 4 hours of Si deficiency. Analysis of the fatty acid composition of the accumulated lipids also showed changes induced by Si starvation. In Si-deficient cells, there was an increase in the proportions of mono- and unsaturated fatty acids (16:1, palmitoleic acid; 16:0, palmitic acid; and 14:0, myristic acid), and a reduction in the proportions of the three major polyunsaturated fatty acids, (16:3, 20:5, and 22:6). These results are consistent with the finding that the predominant

fatty acids found in triacylglycerol storage lipids in C. cryptica are 16:1, 16:0, and 14:1. These

shorter, more highly saturated fatty acids are also the most desirable substrates for conversion into fatty acid methyl esters (biodiesel), as they would be less likely to polymerize during combustion and “gum up” an engine.

Although Si depletion causes all diatoms tested to stop dividing, species responded differently

response to Si-depletion, with a decrease in growth accompanied by a significant increase in the proportion of the biomass as lipid within 12 hours (the response to N starvation was usually much slower, as the cells could utilize internal N stores). This result again emphasizes the need to understand the kinetics of lipid accumulation in individual species under specific conditions for cost-effective lipid production in the ponds.

Figure II.B.3. Changes in lipid mass, ash-free dry mass, and lipid content in Si-deficient cultures of three diatoms.

A. C. fusiformis B. C. cryptica C. T. pseudonana.