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.     

Ocean Warming and CO2-Induced Acidification Impact the Lipid Content of a Marine Predatory Gastropod

Ocean warming and acidification are current global environmental challenges impacting aquatic organisms. A shift in conditions outside the optimal environmental range for marine species is likely to generate stress that could impact metabolic activity, with consequences for the biosynthesis of marine lipids. The aim of this study was to investigate differences in the lipid content of Dicathais orbita exposed to current and predicted future climate change scenarios. The whelks were exposed to a combination of temperature and CO₂-induced acidification treatments in controlled flowthrough seawater mesocosms for 35 days. Under current conditions, D. orbita foot tissue has an average of 6 mg lipid/g tissue, but at predicted future ocean temperatures, the total lipid content dropped significantly, to almost half. The fatty acid composition is dominated by polyunsaturated fatty acids (PUFA 52%) with an n-3:6 fatty acid ratio of almost 2, which remains unchanged under future ocean conditions. However, we detected an interactive effect of temperature and pCO₂ on the % PUFAs and n-3 and n-6 fatty acids were significantly reduced by elevated water temperature, while both the saturated and monounsaturated fatty acids were significantly reduced under increased pCO₂ acidifying conditions. The present study indicates the potential for relatively small predicted changes in ocean conditions to reduce lipid reserves and alter the fatty acid composition of a predatory marine mollusc. This has potential implications for the growth and survivorship of whelks under future conditions, but only minimal implications for human consumption of D. orbita as nutritional seafood are predicted.     

Optimization of Medium Using Response Surface Methodology for Lipid Production by Scenedesmus sp.

Lipid production is an important indicator for assessing microalgal species for biodiesel production. In this work, the effects of medium composition on lipid production by Scenedesmus sp. were investigated using the response surface methodology. The results of a Plackett–Burman design experiment revealed that NaHCO₃, NaH₂PO₄·2H₂O and NaNO₃ were three factors significantly influencing lipid production, which were further optimized by a Box–Behnken design. The optimal medium was found to contain 3.07 g L⁻¹ NaHCO₃, 15.49 mg L⁻¹ NaH₂PO₄·2H₂O and 803.21 mg L⁻¹ NaNO₃. Using the optimal conditions previously determined, the lipid production (304.02 mg·L⁻¹) increased 54.64% more than that using the initial medium, which agreed well with the predicted value 309.50 mg L⁻¹. Additionally, lipid analysis found that palmitic acid (C16:0) and oleic acid (C18:1) dominantly constituted the algal fatty acids (about 60% of the total fatty acids) and a much higher content of neutral lipid accounted for 82.32% of total lipids, which strongly proved that Scenedesmus sp. is a very promising feedstock for biodiesel production.     

Oleosome-Associated Protein of the Oleaginous Diatom Fistulifera solaris Contains an Endoplasmic Reticulum-Targeting Signal Sequence

Microalgae tend to accumulate lipids as an energy storage material in the specific organelle, oleosomes. Current studies have demonstrated that lipids derived from microalgal oleosomes are a promising source of biofuels, while the oleosome formation mechanism has not been fully elucidated. Oleosome-associated proteins have been identified from several microalgae to elucidate the fundamental mechanisms of oleosome formation, although understanding their functions is still in infancy. Recently, we discovered a diatom-oleosome-associated-protein 1 (DOAP1) from the oleaginous diatom, Fistulifera solaris JPCC DA0580. The DOAP1 sequence implied that this protein might be transported into the endoplasmic reticulum (ER) due to the signal sequence. To ensure this, we fused the signal sequence to green fluorescence protein. The fusion protein distributed around the chloroplast as like a meshwork membrane structure, indicating the ER localization. This result suggests that DOAP1 could firstly localize at the ER, then move to the oleosomes. This study also demonstrated that the DOAP1 signal sequence allowed recombinant proteins to be specifically expressed in the ER of the oleaginous diatom. It would be a useful technique for engineering the lipid synthesis pathways existing in the ER, and finally controlling the biofuel quality.     

Lipids and Composition of Fatty Acids of Saccharina latissima Cultivated Year-Round in Integrated Multi-Trophic Aquaculture

This study is evaluating the seasonal lipid and fatty acid composition of the brown seaweed Saccharina latissima. Biomass was sampled throughout the year (bi-monthly) at the commercial cultivation site near a fish farm in an integrated multi-trophic aquaculture (IMTA) and at a reference site in Denmark (2013–2014). Generally, there was no difference in the biomass composition between sites; however, significant seasonal changes were found. The lipid concentration varied from 0.62%–0.88% dry weight (DW) in July to 3.33%–3.35% DW in November (p < 0.05) in both sites. The fatty acid composition in January was significantly different from all the other sampling months. The dissimilarities were mainly explained by changes in the relative abundance of 20:5n-3 (13.12%–33.35%), 14:0 (11.07%–29.37%) and 18:1n-9 (10.15%–16.94%). Polyunsaturated fatty acids (PUFA’s) made up more than half of the fatty acids with a maximum in July (52.3%–54.0% fatty acid methyl esters; FAME). This including the most appreciated health beneficial PUFA’s, eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), but also arachidonic (ARA) and stearidonic acid (SDA), which are not found in land vegetables such as cabbage and lettuce. Compared to fat (salmon) and lean fish (cod) this seaweed species contains higher proportions of ARA and SDA, but lower EPA (only cod) and DHA. Conclusively, the season of harvest is important for the choice of lipid quantity and quality, but the marine vegetables provide better sources of EPA, DHA and long-chain (LC)-PUFA’s in general compared to traditional vegetables.     

Fatty Acid Production and Direct Acyl Transfer through Polar Lipids Control TAG Biosynthesis during Nitrogen Deprivation in the Halotolerant Alga Dunaliella tertiolecta

The aims of this work were to evaluate the contribution of the free fatty acid (FA) pool to triacylglyceride (TAG) biosynthesis and to try to characterize the mechanism by which FA are assimilated into TAG in the green alga Dunaliella tertiolecta. A time-resolved lipidomic analysis showed that nitrogen (N) deprivation induces a redistribution of total lipidome, particularly of free FA and major polar lipid (PL), in parallel to enhanced accumulation of polyunsaturated TAG. The steady-state concentration of the FA pool, measured by prolonged ¹⁴C-bicarbonate pre-labeling, showed that N deprivation induced a 50% decrease in total FA level within the first 24 h and up to 85% after 96 h. The abundance of oleic acid increased from 50 to 70% of total free FA while polyunsaturated FA (PUFA) disappeared under N deprivation. The FA flux, measured by the rate of incorporation of ¹⁴C-palmitic acid (PlA), suggests partial suppression of phosphatidylcholine (PC) acyl editing and an enhanced turnover of the FA pool and of total digalactosyl-diacylglycerol (DGDG) during N deprivation. Taken together, these results imply that FA biosynthesis is a major rate-controlling stage in TAG biosynthesis in D. tertiolecta and that acyl transfer through PL such as PC and DGDG is the major FA assimilation pathway into TAG in that alga and possibly in other green microalgae. Increasing the availability of FA could lead to enhanced TAG biosynthesis and to improved production of high-value products from microalgae.     

High-EPA Biomass from Nannochloropsis salina Cultivated in a Flat-Panel Photo-Bioreactor on a Process Water-Enriched Growth Medium

Nannochloropsis salina was grown on a mixture of standard growth media and pre-gasified industrial process water representing effluent from a local biogas plant. The study aimed to investigate the effects of enriched growth media and cultivation time on nutritional composition of Nannochloropsis salina biomass, with a focus on eicosapentaenoic acid (EPA). Variations in fatty acid composition, lipids, protein, amino acids, tocopherols and pigments were studied and results compared to algae cultivated on F/2 media as reference. Mixed growth media and process water enhanced the nutritional quality of Nannochloropsis salina in laboratory scale when compared to algae cultivated in standard F/2 medium. Data from laboratory scale translated to the large scale using a 4000 L flat panel photo-bioreactor system. The algae growth rate in winter conditions in Denmark was slow, but results revealed that large-scale cultivation of Nannochloropsis salina at these conditions could improve the nutritional properties such as EPA, tocopherol, protein and carotenoids compared to laboratory-scale cultivated microalgae. EPA reached 44.2% ± 2.30% of total fatty acids, and α-tocopherol reached 431 ± 28 µg/g of biomass dry weight after 21 days of cultivation. Variations in chemical compositions of Nannochloropsis salina were studied during the course of cultivation. Nannochloropsis salina can be presented as a good candidate for winter time cultivation in Denmark. The resulting biomass is a rich source of EPA and also a good source of protein (amino acids), tocopherols and carotenoids for potential use in aquaculture feed industry.     

A Saponification Method for Chlorophyll Removal from Microalgae Biomass as Oil Feedstock

Microalgae oil is an optimal feedstock for nutraceutical, pharmaceutical and biodiesel production, but its high levels of chlorophyll limit its large-scale application. To date, few effective approaches have been developed to remove chlorophyll from microalgae oil. The main purpose of this study was to present a preprocessing method of algae oil feedstock (Scenedesmus) to remove chlorophyll by saponification. The results showed that 96% of chlorophyll in biomass was removed. High quality orange transparent oil could be extracted from the chlorophyll reduced biomass. Specifically, the proportion of neutral lipids and saturation levels of fatty acids increased, and the pigments composition became carotenoids-based. The critical parameters of chlorophyll reduced biodiesel conformed to the standards of the USA, China and EU. Sodium copper chlorophyllin could be prepared from the bleaching effluent. The results presented herein offer a useful pathway to improve the quality of microalgae oil and reduce the cost of microalgae biodiesel.     

Heterotrophic Production of Omega-3 Long-Chain Polyunsaturated Fatty Acids by Trophically Converted Marine Diatom Phaeodactylum tricornutum

We have created via metabolic engineering a heterotrophic strain of Phaeodactylum tricornutum that accumulates enhanced levels of the high value omega-3 long chain polyunsaturated fatty acid (LC-PUFAs) docosahexaenoic acid (DHA). This was achieved by generation of transgenic strains in which the Δ5-elongase from Ostreococcus tauri was co-expressed with a glucose transporter from the moss Physcomitrella patens. This double transformant has the capacity to grow in the dark in liquid medium supplemented with glucose and accumulate substantial levels of omega-3 LC-PUFAs. The effects of glucose concentrations on growth and LC-PUFA production of wild type and transformed strains cultivated in the light and dark were studied. The highest omega-3 LC-PUFAs accumulation was observed in cultures grown under mixotrophic conditions in the presence of 1% glucose (up to 32.2% of total fatty acids, TFA). Both DHA and EPA are detected at high levels in the neutral lipids of transgenic cells grown under phototrophic conditions, averaging 36.5% and 23.6% of TFA, respectively. This study demonstrates the potential for P. tricornutum to be developed as a viable commercial strain for both EPA and DHA production under mixo- and heterotrophic conditions.     

Lipid composition and deposition during grain filling in intact barley (Hordeum vulgare) mutant grains as studied by H HR MAS NMR

Conventional barley has an excellent nutritional value, but a lower content of lipids than oat. Nevertheless, barley lipids deserve a focused interest due to their fatty acid composition and their vitamin E composition. In this study, ¹H HR MAS NMR was used to monitor the deposition of lipids during grain filling of conventional barley, and of two barley endosperm mutants with increased lipid content. The lipids in the mutants are primarily synthesized between days 9 and 13, whereas the lipid biosynthesis in the conventional barley took place ten days later. GC analysis on barley flour lipids showed that the mutants and the control exhibit comparable relative levels of the major fatty acids: C16:0, C18:1 and C18:2 despite the higher content of lipids in the mutants. Differences in the minor fatty acid composition between control and mutants were also observed. The degree of unsaturation was found to be increasing during grain filling for both conventional barley and the two mutants.     

Multiple Beneficial Lipids Including Lecithin Detected in the Edible Invasive Mollusk Crepidula fornicata from the French Northeastern Atlantic Coast

The invasive mollusk Crepidula fornicata, occurring in large amounts in bays along the French Northeastern Atlantic coasts, may have huge environmental effects in highly productive ecosystems where shellfish are exploited. The present study aims at determining the potential economic value of this marine species in terms of exploitable substances with high added value. Lipid content and phospholipid (PL) composition of this mollusk collected on the Bourgneuf Bay were studied through four seasons. Winter specimens contained the highest lipid levels (5.3% dry weight), including 69% of PLs. Phosphatidylcholine (PC) was the major PL class all year, accounting for 63.9% to 88.9% of total PLs. Consequently, the winter specimens were then investigated for PL fatty acids (FAs), and free sterols. Dimethylacetals (DMAs) were present (10.7% of PL FA + DMA mixture) revealing the occurrence of plasmalogens. More than forty FAs were identified, including 20:5n-3 (9.4%) and 22:6n-3 (7.3%) acids. Fourteen free sterols were present, including cholesterol at 31.3% of the sterol mixture and about 40% of phytosterols. These data on lipids of C. fornicata demonstrate their positive attributes for human nutrition and health. The PL mixture, rich in PC and polyunsaturated FAs, offers an interesting alternative source of high value-added marine lecithin.     

Improvement of Neutral Lipid and Polyunsaturated Fatty Acid Biosynthesis by Overexpressing a Type 2 Diacylglycerol Acyltransferase in Marine Diatom Phaeodactylum tricornutum

Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the last step of triacylglyceride (TAG) biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.     

Distinguishing Health Benefits of Eicosapentaenoic and Docosahexaenoic Acids

Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) are recommended for management of patients with wide-ranging chronic diseases, including coronary heart disease, rheumatoid arthritis, dementia, and depression. Increased consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is recommended by many health authorities to prevent (up to 0.5 g/day) or treat chronic disease (1.0 g/day for coronary heart disease; 1.2–4 g/day for elevated triglyceride levels). Recommendations for dietary intake of LC n-3 PUFAs are often provided for α-linolenic acid, and for the combination of EPA and DHA. However, many studies have also reported differential effects of EPA, DHA and their metabolites in the clinic and at the laboratory bench. The aim of this article is to review studies that have identified divergent responses to EPA and DHA, and to explore reasons for these differences. In particular, we review potential contributing factors such as differential membrane incorporation, modulation of gene expression, activation of signaling pathways and metabolite formation. We suggest that there may be future opportunity to refine recommendations for intake of individual LC n-3 PUFAs.     

Nutritional and Chemical Composition and Antiviral Activity of Cultivated Seaweed Sargassum naozhouense Tseng et Lu

Sargassum naozhouense is a brown seaweed used in folk medicine and applied for thousands of years in Zhanjiang, Guangdong province, China. This study is the first time to investigate its chemical composition and antiviral activity. On the dry weight basis, this seaweed was constituted of ca. 35.18% ash, 11.20% protein, 1.06% lipid and 47.73% total carbohydrate, and the main carbohydrate was water-soluble polysaccharide. The protein analysis indicated the presence of essential amino acids, which accounted for 36.35% of the protein. The most abundant fatty acids were C14:0, C16:0, C18:1 and C20:4. The ash fraction analysis indicated that essential minerals and trace elements, such as Fe, Zn and Cu, were present in the seaweed. IR analysis revealed that polysaccharides from cultivated S. naozhouense may be alginates and fucoidan. The polysaccharides possessed strong antiviral activity against HSV-1 in vitro with EC₅₀ of 8.92 μg/mL. These results demonstrated cultivated S. naozhouense has a potential for its use in functional foods and antiviral new drugs.     

Combined Effects of Nitrogen Concentration and Seasonal Changes on the Production of Lipids in Nannochloropsis oculata

Instead of sole nutrient starvation to boost algal lipid production, we addressed nutrient limitation at two different seasons (autumn and spring) during outdoor cultivation in flat panel photobioreactors. Lipid accumulation, biomass and lipid productivity and changes in fatty acid composition of Nannochloropsis oculata were investigated under nitrogen (N) limitation (nitrate:phosphate N:P 5, N:P 2.5 molar ratio). N. oculata was able to maintain a high biomass productivity under N-limitation compared to N-sufficiency (N:P 20) at both seasons, which in spring resulted in nearly double lipid productivity under N-limited conditions (0.21 g L⁻¹ day⁻¹) compared to N-sufficiency (0.11 g L⁻¹ day⁻¹). Saturated and monounsaturated fatty acids increased from 76% to nearly 90% of total fatty acids in N-limited cultures. Higher biomass and lipid productivity in spring could, partly, be explained by higher irradiance, partly by greater harvesting rate (~30%). Our results indicate the potential for the production of algal high value products (i.e., polyunsaturated fatty acids) during both N-sufficiency and N-limitation. To meet the sustainability challenges of algal biomass production, we propose a dual-system process: Closed photobioreactors producing biomass for high value products and inoculum for larger raceway ponds recycling waste/exhaust streams to produce bulk chemicals for fuel, feed and industrial material.     

Characterization of Shrimp Oil from Pandalus borealis by High Performance Liquid Chromatography and High Resolution Mass Spectrometry

Northern shrimp (Pandalus borealis) oil, which is rich in omega-3 fatty acids, was recovered from the cooking water of shrimp processing facilities. The oil contains significant amounts of omega-3 fatty acids in triglyceride form, along with substantial long-chain monounsaturated fatty acids (MUFAs). It also features natural isomeric forms of astaxanthin, a nutritional carotenoid, which gives the oil a brilliant red color. As part of our efforts in developing value added products from waste streams of the seafood processing industry, we present in this paper a comprehensive characterization of the triacylglycerols (TAGs) and astaxanthin esters that predominate in the shrimp oil by using HPLC-HRMS and MS/MS, as well as ¹³C-NMR. This approach, in combination with FAME analysis, offers direct characterization of fatty acid molecules in their intact forms, including the distribution of regioisomers in TAGs. The information is important for the standardization and quality control, as well as for differentiation of composition features of shrimp oil, which could be sold as an ingredient in health supplements and functional foods.     

Alternative Sources of n-3 Long-Chain Polyunsaturated Fatty Acids in Marine Microalgae

The main source of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) in human nutrition is currently seafood, especially oily fish. Nonetheless, due to cultural or individual preferences, convenience, geographic location, or awareness of risks associated to fatty fish consumption, the intake of fatty fish is far from supplying the recommended dietary levels. The end result observed in most western countries is not only a low supply of n-3 LC-PUFA, but also an unbalance towards the intake of n-6 fatty acids, resulting mostly from the consumption of vegetable oils. Awareness of the benefits of LC-PUFA in human health has led to the use of fish oils as food supplements. However, there is a need to explore alternatives sources of LC-PUFA, especially those of microbial origin. Microalgae species with potential to accumulate lipids in high amounts and to present elevated levels of n-3 LC-PUFA are known in marine phytoplankton. This review focuses on sources of n-3 LC-PUFA, namely eicosapentaenoic and docosahexaenoic acids, in marine microalgae, as alternatives to fish oils. Based on current literature, examples of marketed products and potentially new species for commercial exploitation are presented.     

Changes in phytochemical, antimicrobial and free radical scavenging activities of the Peruvian pepper tree (Schinus molle L.) as influenced by fruit maturation

The effect of stage of maturity on total lipids, fatty acids, yields and essential oil composition and their antimicrobial and free radical scavenging activities of the Schinus molle fruits was investigated. The content of total lipids varied from 2.87 to 5.35% (w/w, dw) and were rich in unsaturated fatty acids particularly linoleic acid. As maturation progress, the essential oil yield dropped from 5.18% to 1.15%. Monoterpene hydrocarbons with α-phellandrene (35.15-40.38%), limonene + β-phellandrene (21.47-36.62%), β-myrcene (7.61-24.96%) and α-pinene (1.92-2.58%) were found to be the main components. At the same time, the essential oils were evaluated for their antimicrobial and free radical scavenging activities. They were found to be active against Bacillus subtilis, Bacillus cereus, Salmonella typhymurium and Escherichia coli but they do not inhibit the growth of Candida albicans. Conversely, they showed very weak activity against the DPPH radical. In both assay, the oil derived from the intermediate stage was reported as more efficient.