Freddy Guihéneuf

LC-PUFA-Enriched Oil Production by Microalgae: Accumulation of Lipid and Triacylglycerols Containing n-3 LC-PUFA Is Triggered by Nitrogen Limitation and Inorganic Carbon Availability in the Marine Haptophyte Pavlova lutheri

In most microalgal species, triacyglycerols (TAG) contain mostly saturated and monounsaturated fatty acids, rather than PUFA, while PUFA-enriched oil is the form most desirable for dietary intake. The ability of some species to produce LC-PUFA-enriched oil is currently of specific interest. In this work, we investigated the role of sodium bicarbonate availability on lipid accumulation and n-3 LC-PUFA partitioning into TAG during batch cultivation of Pavlova lutheri. Maximum growth and nitrate uptake exhibit an optimum concentration and threshold tolerance to bicarbonate addition (~9 mM) above which both parameters decreased. Nonetheless, the transient highest cellular lipid and TAG contents were obtained at 18 mM bicarbonate, immediately after combined alkaline pH stress and nitrate depletion (day nine), while oil body and TAG accumulation were highly repressed with low carbon supply (2 mM). Despite decreases in the proportions of EPA and DHA, maximum volumetric and cellular EPA and DHA contents were obtained at this stage due to accumulation of TAG containing EPA/DHA. TAG accounted for 74% of the total fatty acid per cell, containing 55% and 67% of the overall cellular EPA and DHA contents, respectively. These results clearly demonstrate that inorganic carbon availability and elevated pH represent two limiting factors for lipid and TAG accumulation, as well as n-3 LC-PUFA partitioning into TAG, under nutrient-depleted P. lutheri cultures.

The Anti-Inflammatory Effect of Algae-Derived Lipid Extracts on Lipopolysaccharide (LPS)-Stimulated Human THP-1 Macrophages

Algae contain a number of anti-inflammatory bioactive compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) and chlorophyll a, hence as dietary ingredients, their extracts may be effective in chronic inflammation-linked metabolic diseases such as cardiovascular disease. In this study, anti-inflammatory potential of lipid extracts from three red seaweeds (Porphyra dioica, Palmaria palmata and Chondrus crispus) and one microalga (Pavlova lutheri) were assessed in lipopolysaccharide (LPS)-stimulated human THP-1 macrophages. Extracts contained 34%–42% total fatty acids as n-3 PUFA and 5%–7% crude extract as pigments, including chlorophyll a, β-carotene and fucoxanthin. Pretreatment of the THP-1 cells with lipid extract from P. palmata inhibited production of the pro-inflammatory cytokines interleukin (IL)-6 (p < 0.05) and IL-8 (p < 0.05) while that of P. lutheri inhibited IL-6 (p < 0.01) production. Quantitative gene expression analysis of a panel of 92 genes linked to inflammatory signaling pathway revealed down-regulation of the expression of 14 pro-inflammatory genes (TLR1, TLR2, TLR4, TLR8, TRAF5, TRAF6, TNFSF18, IL6R, IL23, CCR1, CCR4, CCL17, STAT3, MAP3K1) by the lipid extracts. The lipid extracts effectively inhibited the LPS-induced pro-inflammatory signaling pathways mediated via toll-like receptors, chemokines and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling molecules. These results suggest that lipid extracts from P. lutheri, P. palmata, P. dioica and C. crispus can inhibit LPS-induced inflammatory pathways in human macrophages. Therefore, algal lipid extracts should be further explored as anti-inflammatory ingredients for chronic inflammation-linked metabolic diseases.

ENVIRONMENTAL FACTORS AFFECTING GROWTH AND OMEGA 3 FATTY ACID COMPOSITION INSKELETONEMA COSTATUM.THE INFLUENCES OF IRRADIANCE AND CARBON SOURCE: Communication presented at the 25èmeCongrès Annuel de l'Association des Diatomistes de Langue Francaise (ADLaF), Caen, 25-28 September 2006

The effects of carbon sources and three different irradiance levels on the growth and polyunsaturated fatty acid composition of the diatom Skeletonema costatum have been studied. Diatoms were grown in artificial seawater (ASW), which contained sodium bicarbonate (photoautotrophic growth) or sodium acetate (photoheterotrophic growth) at the same concentration (2.07 mM). The optimum growth conditions were between 100–340 μmol photons m−2 s−1 in presence of sodium bicarbonate, both of which enhance photosynthesis. However, under limiting irradiance, results suggested that sodium acetate was more efficiently used than sodium bicarbonate during batch growth. As a consequence, in natural conditions and under low irradiance, availability of sodium acetate could be a limiting factor for cell metabolism. This diatom also seems to be able to support efficiently mixotrophic growth under low irradiance (20 μmol photons m−2 s−1). The highest eicosapentaenoic acid (EPA) amounts were observed when cells were cultured under saturating irradiance (340 μmol photons m−2 s−1) during the late experimental and stationary phases. On the contrary, an increase in the EPA level was observed during the decline phase under limiting irradiance. Indeed, at low irradiance, an accumulation of omega 3 fatty acids in the thylakoid membranes could facilitate the photosynthetic reactions suggesting a peculiar photoadaptation of Skeletonema costatum. Finally, during the exponential phase, a decreased of omega 3 fatty acid amount and specifically EPA with acetate as a carbon source has been observed under limiting and saturating irradiance. This could be explained by the decreased acetate availability that could be considered as responsible for the fall in EPA level.The effects of carbon sources and three different irradiance levels on the growth and polyunsaturated fatty acid composition of the diatom Skeletonema costatum have been studied. Diatoms were grown in artificial seawater (ASW), which contained sodium bicarbonate (photoautotrophic growth) or sodium acetate (photoheterotrophic growth) at the same concentration (2.07 mM). The optimum growth conditions were between 100–340 μmol photons m−2 s−1 in presence of sodium bicarbonate, both of which enhance photosynthesis. However, under limiting irradiance, results suggested that sodium acetate was more efficiently used than sodium bicarbonate during batch growth. As a consequence, in natural conditions and under low irradiance, availability of sodium acetate could be a limiting factor for cell metabolism. This diatom also seems to be able to support efficiently mixotrophic growth under low irradiance (20 μmol photons m−2 s−1). The highest eicosapentaenoic acid (EPA) amounts were observed when cells were cultured un...

Genetic Engineering: A Promising Tool to Engender Physiological, Biochemical, and Molecular Stress Resilience in Green Microalgae

As we march into the 21st century, the prevailing scenario of depleting energy resources, global warming and ever increasing issues of human health and food security will quadruple. In this context, genetic and metabolic engineering of green microalgae complete the quest toward a continuum of environmentally clean fuel and food production. Evolutionarily related, but unlike land plants, microalgae need nominal land or water, and are best described as unicellular autotrophs using light energy to fix atmospheric carbon dioxide (CO2) into algal biomass, mitigating fossil CO2 pollution in the process. Remarkably, a feature innate to most microalgae is synthesis and accumulation of lipids (60–65% of dry weight), carbohydrates and secondary metabolites like pigments and vitamins, especially when grown under abiotic stress conditions. Particularly fruitful, such an application of abiotic stress factors such as nitrogen starvation, salinity, heat shock, etc., can be used in a biorefinery concept for production of multiple valuable products. The focus of this mini-review underlies metabolic reorientation practices and tolerance mechanisms as applied to green microalgae under specific stress stimuli for a sustainable pollution-free future. Moreover, we entail current progress on genetic engineering as a promising tool to grasp adaptive processes for improving strains with potential biotechnological interests.

Light-dependent utilization of two radiolabelled carbon sources, sodium bicarbonate and sodium acetate, and relationships with long chain polyunsaturated fatty acid synthesis in the microalga Pavlova lutheri (Haptophyta)

Pavlova lutheri is a common member of the Pavlovophyceae (Haptophyta), often used as a food source for aquatic filter-feeders and cultured in laboratories to produce high levels of polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively), which are known to have benefits for human health. Consequently, we investigated the pathways involved in the biosynthesis of long chain polyunsaturated fatty acids (LC-PUFAs) in this alga during photosynthesis in relation to light intensity. Using two radiolabelled carbon sources, [14C] sodium bicarbonate and [1-14C] sodium acetate, we obtained data suggesting that P. lutheri is able to synthesize LC-PUFAs by successive elongation and desaturation steps. It converts palmitic acid into palmitoleic acid by Δ7-desaturation. Moreover, significant incorporation of [1-14C] acetate (organic carbon) and its subsequent use in lipid metabolism suggest that P. lutheri may have a mixotrophic capacity for carbon assimilation. Synthesis of lipids, including galactolipids and phospholipids, increased with light intensity when the cells were incubated with [14C] bicarbonate (inorganic carbon), but was less sensitive to differences in light intensity when incubated with [1-14C] acetate, a heterotrophic carbon source that stimulates the synthesis of monounsaturated fatty acids, such as oleic acid. In the case of n-3 fatty acids, EPA and DHA synthesis was lower at high light (340 µmol photons m−2 s−1) with the [14C] bicarbonate, but did not vary with [1-14C] acetate. Finally, P. lutheri seems to have two distinct enzyme pools involved in LC-PUFA synthesis, one is intra-chloroplastidic and dependent on light intensity, and the other is extra-chloroplastidic and independent of light.

Seasonal and geographical variations in the biochemical composition of the blue mussel (Mytilus edulis L.) from Ireland.

Blue mussel (Mytilus edulis L.) farming constitutes the largest volume of the shellfish sector in Ireland. Recently, interest in mussel dietary supplements and functional foods has increased significantly. To identify the optimal harvesting time and location in Ireland, blue mussels were investigated for their biochemical composition over a period of one year. The study included samples from aquaculture facilities, wild grown mussels and waste material. Each sample was analysed at four time points to determine the total content of (i) glycogen, (ii) lipids, (iii) proteins, (iv) inorganic substances, and (v) energy. Moreover, fatty acid profiles were investigated by GC-FID revealing high contents of PUFAs and a high ω-3/ω-6 ratio. Compared to less pronounced geographical variations, distinct seasonal trends could be observed for all samples. The content of the investigated metabolite classes, inorganic substances, and energy was at a maximum level in spring or late summer.

Light Intensity Regulates LC-PUFA Incorporation into Lipids of Pavlova lutheri and the Final Desaturase and Elongase Activities Involved in Their Biosynthesis.

The microalga Pavlova lutheri is a candidate for the production of omega-3 long-chain polyunsaturated fatty acid (LC-PUFA), due to its ability to accumulate both eicosapentaenoic (EPA) and docosahexaenoic acids. Outstanding questions need to be solved to understand the complexity of n-3 LC-PUFA synthesis and partitioning into lipids, especially its metabolic regulation, and which enzymes and/or abiotic factors control their biosynthesis. In this study, the radioactivity of 14C-labeled arachidonic acid incorporated into the total lipids of P. lutheri grown under different light intensities and its conversion into labeled LC-PUFA were monitored. The results highlighted for the first time the light-dependent incorporation of LC-PUFA into lipids and the light-dependent activity of the final desaturation and elongation steps required to synthesize and accumulate n-3 C20/C22 LC-PUFA. The incorporation of arachidonic acid into lipids under low light and the related Δ17-desaturation activity measured explain the variations in fatty acid profile of P. lutheri, especially the accumulation of n-3 LC-PUFA such as EPA under low light conditions.

Evaluation of food grade solvents for lipid extraction and impact of storage temperature on fatty acid composition of edible seaweeds Laminaria digitata (Phaeophyceae) and Palmaria palmata (Rhodophyta).

This study evaluated the impact of different food- and non-food grade extraction solvents on yield and fatty acid composition of the lipid extracts of two seaweed species (Palmaria palmata and Laminaria digitata). The application of chloroform/methanol and three different food grade solvents (ethanol, hexane, ethanol/hexane) revealed significant differences in both, extraction yield and fatty acid composition. The extraction efficiency, in terms of yields of total fatty acids (TFA), was in the order: chloroform/methanol>ethanol>hexane>ethanol/hexane for both species. Highest levels of polyunsaturated fatty acids (PUFA) were achieved by the extraction with ethanol. Additionally the effect of storage temperature on the stability of PUFA in ground and freeze-dried seaweed biomass was investigated. Seaweed samples were stored for a total duration of 22months at three different temperatures (-20°C, 4°C and 20°C). Levels of TFA and PUFA were only stable after storage at -20°C for the two seaweed species.

Lipids and Fatty Acids in Algae: Extraction, Fractionation into Lipid Classes, and Analysis by Gas Chromatography Coupled with Flame Ionization Detector (GC-FID)

Despite the number of biochemical studies exploring algal lipids and fatty acid biosynthesis pathways and profiles, analytical methods used by phycologists for this purpose are often diverse and incompletely described. Potential confusion and potential variability of the results between studies can therefore occur due to change of protocols for lipid extraction and fractionation, as well as fatty acid methyl esters (FAME) preparation before gas chromatography (GC) analyses. Here, we describe a step-by-step procedure for the profiling of neutral and polar lipids using techniques such as solid-liquid extraction (SLE), thin-layer chromatography (TLC), and gas chromatography coupled with flame ionization detector (GC-FID). As an example, in this protocol chapter, analyses of neutral and polar lipids from the marine microalga Pavlova lutheri (an EPA/DHA-rich haptophyte) will be outlined to describe the distribution of fatty acid residues within its major lipid classes. This method has been proven to be a reliable technique to assess changes in lipid and fatty acid profiles in several other microalgal species and seaweeds.

Effects of an experimental heat wave on fatty acid composition in two Mediterranean seagrass species

Global warming is emerging as one of the most critical threats to terrestrial and marine species worldwide. This study assessed the effects of simulated warming events in culture on two seagrass species, Posidonia oceanica and Cymodocea nodosa, which play a key role in coastal ecosystems of the Mediterranean Sea. Changes in fatty acids as key metabolic indicators were assessed in specimens from two geographical populations of each species adapted to different in situ temperature regimes. Total fatty acid (TFA) content and composition were compared in C. nodosa and P. oceanica from natural populations and following exposure to heat stress in culture. After heat exposure, individuals of C. nodosa and P. oceanica adapted to colder temperatures in situ accumulated significantly more TFA than controls. For both species, the proportion of polyunsaturated fatty acids (PUFA) decreased, and the percentage of saturated fatty acids (SFA) increased significantly after the heat treatment. These results highlight that populations of both species living at warmest temperatures in situ were more thermo-tolerant and exhibited a greater capacity to cope with heat stress by readjusting their lipid composition faster. Finally, exposure of seagrasses to warmer conditions may induce a decrease in PUFA/SFA ratio which could negatively affect their nutritional value and generate important consequences in the healthy state of next trophic levels.