We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics.lipid analysis | bioenergy T he global concerns surrounding unabated fossil fuel consumption and the risk of significant environmental impact caused by the associated greenhouse gas emissions, compounded by potential challenges associated with land-based biofuels, have renewed significant interest in microalgae as an alternative feedstock for the production of biodiesel and other biofuels (1). Microalgae hold considerable promise because of their ability to synthesize and store lipids, such as fatty acids and triacylglycerols (TAGs), which can be readily converted into biodiesel (fatty acid methyl or ethyl esters) through relatively simple chemical reactions (2). Small yet efficient, microalgae are attractive for many reasons, including their rapid, cost-effective, and resource-efficient production on nonarable land or photobioreactors (3), with impaired water, and for especially significant lipid production-up to 20-50% of their total dry weight, with examples of up to 80% under certain conditions reported (4). It has been estimated that lipid production of microalgae could be 30 times more efficient in terms of relative production of lipids per acre per year than any other terrestrial plant oil feedstock (2, 5).Under optimal growth conditions, microalgae synthesize fatty acids in the form of various glycerol-based membrane lipids primarily for structural and functional roles (6). In contrast, adverse environmental or metabolic stress conditions such as nutrient limitation, commonly referred to as "lipid trigger" conditions, result in an increase in carbon partitioning and accumulation of substantial proportions of neutral lipids (20-50% of dry weight), primarily in the form of TAGs. The TAGs are a form of...

No abstract

No abstract

No abstract

The Center for Biostabilization at UC Davis is attempting to stabilize mammalian cells in the dry state. We review here some of the lessons from nature that we have been applying to this enterprise, including the use of trehalose, a disaccharide found at high concentrations in many anhydrobiotic organisms, to stabilize biological structures, both in vitro and in vivo. Trehalose has useful properties for this purpose and in at least in one case-human blood platelets-introducing this sugar may be sufficient to achieve useful stabilization. Nucleated cells, however, are stabilized by trehalose only during the initial stages of dehydration. Introduction of a stress protein obtained from an anhydrobiotic organism, Artemia, improves the stability markedly, both during the dehydration event and following rehydration. Thus, it appears that the stabilization will require multiple adaptations, many of which we propose to apply from studies on anhydrobiosis.

We have investigated raft formation in human platelets in response to cell activation. Lipid phase separation and domain formation were detected using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (diI-C(18)) that preferentially partitions into gel-like lipid domains. We showed that when human platelets are activated by cold and physiological agonists, rafts coalesce into visible aggregates. These events were disrupted by depletion of membrane cholesterol. Using Fourier transform infrared spectroscopy (FTIR), we measured a thermal phase transition at around 30 degrees C in intact platelets, which we have assigned as the liquid-ordered to the liquid-disordered phase transition of rafts. Phase separation of the phospholipid and the sphingomyelin-enriched rafts could be observed as two phase transitions at around 15 and 30 degrees C, respectively. The higher transition, assigned to the rafts, was greatly enhanced with removal of membrane cholesterol. Detergent-resistant membranes (DRMs) were enriched in cholesterol (50%) and sphingomyelin (20%). The multi-functional platelet receptor CD36 selectively partitioned into DRMs, whereas the GPI-linked protein CD55 and the major platelet integrin alpha(IIb)beta(3a) did not, which suggests that the clustering of proteins within rafts is a regulated process dependent on specific lipid protein interactions. We suggest that raft aggregation is a dynamic, reversible physiological event triggered by cell activation.

Anhydrobiosis, or life without water, is the remarkable ability of certain types of plants and animals to survive almost total dehydration. This phenomenon requires a coordinated series of events within the cells of anhydrobiotes that protect their cellular components, particularly proteins and lipid membranes, from damage caused by the removal of water. Much of what is now understood about preserving biological samples during drying was learned by studying naturally desiccation-tolerant organisms and extended using model systems such as phospholipid vesicles. Most anhydrobiotic organisms accumulate disaccharides in their cells and tissues during the dehydration process. These carbohydrates, usually sucrose or trehalose, satisfy two criteria that appear to be necessary for protecting membranes during desiccation and during storage in the dry state. These requirements include: (1) depression of the gel-to-liquid crystalline phase transition temperature (Tm) in the dehydrated lipid to a temperature at or near that of the hydrated lipid, a process that appears to require a direct interaction between the carbohydrates and the lipid molecules of the membrane; and (2) formation of a carbohydrate glass with a relatively high glass transition temperature, leading to inhibition of fusion between the vesicles.

No abstract