Production of eicosapentaenoic acid (EPA) in Monodus subterraneus grown in a helical tubular photobioreactor as affected by cell density and light intensity

The effect of cell density (1–4.5 g L^-1) and light intensity (44 and 82 mol m^-2 s^-1) on fatty acid composition andeicosapentaenoic acid (EPA, 20:5 3) production was studied ina semi-continuous culture of Monodus subterraneus grown in a helicaltubular photobioreactor (`Biocoil') under laboratory conditions. Under lowlight, the highest proportion of EPA (31.5% of total fatty acids) and EPAcontent (3.5% of dry weight), biomass productivity (1.3 g L^-124 h^-1) and EPA productivity (44 mg L^-1 24 h^-1)occurred at optimal cell density of about 1.7 g L^-1. Cell densityhad no effect on the total fatty acid (TFA) content and was maintained atca. 11% of dry weight. Under high light, the highest proportion ofEPA to fatty acids (31.8%), the total fatty acids content (13.4%) andEPA content (4.3% of dry weight) occurred at cell density of about 3.4gL^-1. But the highest biomass productivity (1.7 g L^-124 h^-1) and EPA productivity (56 mg L^-1 24 h^-1) wereobtained at a cell density of 1.6 and 2.6g L^-1, respectively. Ourresults suggest that manipulating the cell density and light intensity canmodify the composition of fatty acid and production of eicosapentaenoicacid (EPA) in M. subterraneus.     

Oil and eicosapentaenoic acid production by the diatom Phaeodactylum tricornutum cultivated outdoors in Green Wall Panel (GWP®) reactors

Phaeodactylum tricornutum is a widely studied diatom and has been proposed as a source of oil and polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic acid (EPA). Recent studies indicate that lipid accumulation occurs under nutritional stress. Aim of this research was to determine how changes in nitrogen availability affect productivity, oil yield, and fatty acid (FA) composition of P. tricornutum UTEX 640. After preliminary laboratory trials, outdoor experiments were carried out in 40‐L GWP® reactors under different nitrogen regimes in batch. Nitrogen replete cultures achieved the highest productivity of biomass (about 18 g m^?2 d^?1) and EPA (about 0.35 g m^?2 d^?1), whereas nitrogen‐starved cultures achieved the highest FA productivity (about 2.6 g m^?2 d^?1). The annual potential yield of P. tricornutum grown outdoors in GWP® reactors is 730 kg of EPA per hectare under nutrient‐replete conditions and 5,800 kg of FA per hectare under nitrogen starvation. Biotechnol. Bioeng. 2017;114: 2204–2210. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

氮源种类及浓度对眼点拟微绿球藻生长密度、油脂产率和二十碳五烯酸含量的影响

氮源是影响微藻生长和油脂积累的重要因素,文中通过单因素试验比较了NaNO3、CO(NH2)2、NH4Cl、CH3COONH4及其浓度对眼点拟微绿球藻生长密度、生长速率、油脂产率、二十碳五烯酸(EPA)含量的影响。结果表明：NH4+更易被眼点拟微绿球藻利用,能更好地促进微藻生长和油脂积累;氮浓度的增加有利于微藻的生长和藻油脂肪酸的去饱和,但不利于微藻油脂的积累。在实验考察的氮源种类和浓度范围内,CH3COONH4是促进眼点拟微绿球藻生长和油脂积累、EPA生成的适宜氮源,其适宜的浓度为5.29 mmol/L。     

Increased production of eicosapentaenoic acid by Skeletonema costatum cells after decantation at low temperature

Coagulation of Skeletonema costatum biomass was induced at alkaline pH in enriched sea-water to concentrate the microalgal suspensions by decantation. Incubation of the concentrated cultures at 15 °C after return at pH 7.5 preserved cell viability and increased the yield of lipid extraction and the 20:5(n-3) fatty acid content of the diatom biomass.     

Oleate and eicosapentaenoic acid attenuate palmitate-induced inflammation and apoptosis in renal proximal tubular cell

Free fatty acid (FFA)-bound albumin, which is filtrated through the glomeruli and reabsorbed into proximal tubular cells, is one of the crucial mediators of tubular damage in proteinuric kidney disease. In this study, we examined the role of each kind of FFA on renal tubular damage in vitro and tried to identify its molecular mechanism. In cultured proximal tubular cells, a saturated fatty acid, palmiate, increased the expression of monocyte chemoattractant protein-1 (MCP-1), but this effect was abrogated by co-incubation of monounsaturated fatty acid, oleate, or ω-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA). Palmitate led to intracellular accumulation of diacylglycerol (DAG) and subsequent activation of protein kinase C protein family. Among the several PKC inhibitors, rottlerin, a PKCθ inhibitor, prevented palmitate-induced MCP-1 expression via inactivation of NFB pathway. Overexpression of dominant-negative PKCθ also inhibited palmitate-induced activation of MCP-1 promoter. Furthermore, palmitate enhanced PKCθ-dependent mitochondrial apoptosis, which was also prevented by co-incubation with oleate or EPA through restoration of pro-survival Akt pathway. Moreover, oleate and EPA inhibited palmitate-induced PKCθ activation through the conversion of intracellular DAG to triglyceride with the restoration of diacylglycerol acyltransferase 2 expression. These results suggest that oleate and EPA have protective effects against the palmitate-induced renal tubular cell damage by inhibiting PKCθ activation.     

Lipase-catalyzed glycerolysis of an oil rich in eicosapentaenoic acid residues

The activities of four immobilized lipases for glycerolysis of a commercially available fish oil (TG500) rich in eicosapentaenoic residues (>58%, w/w) have been characterized in solvent-free systems. The effects of the mole ratio of TG500 to glycerol and temperature have been investigated. The highest conversion was obtained at 60°C with a Candida antarctica fraction B lipase (Chirazyme L-2) and a mole ratio of TG500 (based on fatty acid equivalents) to glycerol of 1.5 to 1.     

Effect of exogenous fatty acids on growth,membrane fluidity,and phospholipid fatty acid composition in yeast

The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-Δ¹¹- eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 1 5:0, and 1 6:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.     

有机碳源对三角褐指藻生长、胞内物质和脂肪酸组分的影响

研究了3种有机碳对三角褐指藻生长、胞内物质和脂肪酸组分的影响。结果表明, 三角褐指藻具有利用有机碳进行兼养生长的能力, 生长速率加快, 倍增时间缩短, 生物量显著提高, 100 mmol/L甘油兼养的生物量最高(713 mg/L), 是自养(460 mg/L)的1.60倍, 乙酸钠和葡萄糖兼养的生物量分别是自养的1.28倍和1.21倍。兼养下蛋白质含量较自养明显下降, 碳水化合物和总脂含量高于自养, 乙酸钠和甘油兼养的总脂含量分别是自养的1.43倍和1.20倍, 葡萄糖兼养的总脂含量与自养无明显差异。3种有机碳兼养的饱和脂肪酸和单不饱和脂肪酸占总脂肪酸的比例增大, 多不饱和脂肪酸比例降低, EPA(eicosapentaenoic acid)比例降低, 乙酸钠兼养的胞内EPA含量(6.23%)和产量(36.59 mg/L)均高于自养, 分别是自养的1.10倍和1.40倍, 甘油和葡萄糖兼养的EPA含量和产量均低于自养。     

Pressurized extraction of unsaturated fatty acids and carotenoids from wet Chlorella vulgaris and Phaeodactylum tricornutum biomass using subcritical liquids

The objective of this study was to investigate the extraction of lipids, for example, mono‐ and polyunsaturated fatty acids (PUFA) as well as carotenoids, from wet microalgae biomass using pressurized subcritical extraction solvents, which meet the requirements of food and feed applications. To demonstrate the effect of the solvent and temperature on the lipid yield, we chose two microalgae species, viz. Chlorella vulgaris and Phaeodactylum tricornutum, differing in their biochemical composition fundamentally. In case of P. tricornutum, ethanol showed the highest fatty acid yield of 85.9% w/w. In addition to eicosapentaenoic acid (EPA), the ethanolic extracts contained exceptional amounts of fucoxanthin (up to 26.1 mg/g d. w.), which can be beneficial to protect unsaturated fatty acids from oxidation processes and in terms of human nutrition. For C. vulgaris, a fatty acid yield of 76.5% w/w was achieved from wet biomass using ethyl acetate at 150°C. In general, an increase in the extraction temperature up to 150°C was found to be important in terms of fatty acid yield when extracting wet microalgae biomass. The results suggest that it is possible to efficiently extract both fatty acids and carotenoids from wet microalgae by selecting suitable solvents and thus circumvent energy‐intensive drying of the biomass.     

Fatty acid composition of a new marine picoplankton species of the Chromophyta

A marine yellowish picoplankton, strain PP301, which was newly isolated from the surface seawater of the western Pacific Ocean was an eminent producer of polyunsaturated fatty acids. Its fatty acids were mostly shared by the shortest saturated form (14:0, 20–30%) and polyunsaturated forms (20:4, EPA and DHA) which accounted for about 50% of the total fatty acids. The amount of intermediate forms in 16 and 18 carbon chains were very little. This composition was consistently observed irrespective of the growth temperatures (15–35 °C).     

Effect of cultural conditions on production of eicosapentaenoic acid byPythium irregulare

Summary The effect of culture conditions upon lipid content and fatty acid composition of mycelia ofPythium irregulare was investigated with particular attention to increasing the yield of 5,8,11,14,17-eicosapentaenoic acid (205; –3) (EPA). All experiments were done by shake flask culture using a yeast extract + malt extract medium. The maximum growth rate was obtained at 25°C, but maximum EPA production was obtained at 12°C. The highest EPA production was 76.5 g EPA/ml 13 days fermentation at 12°C. Addition of glucose during fermentation increased the yield considerably. The highest yield was 112 g/ml, obtained at 13 days fermentation with spiking on day 11. Fermentation time could be shortened by initial incubation at 25°C for 2 days, followed by incubation at 12°C for 6 days. The culture also produced arachidonic acid and other -6 polyunsaturated fatty acids. EPA production was also obtained with lactose or sweet whey permeate, a by-product of cheese manufacture that contains lactose as the main carbohydrate.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Taxonomic differences in the essential fatty acid composition of groups of freshwater zooplankton relate to reproductive demands and generation time

1. Marked differences were observed in the total fatty acid concentrations and essential fatty acid (EFA) distributions of co-existing freshwater copepods and cladocerans in four large lake systems (lakes Michigan, Erie, Ontario and Champlain) over two growing seasons. These patterns appeared independent of lake seston EFA composition.
2. Compared to the cladocerans, calanoid and cyclopoid copepods contained significantly higher concentrations of total fatty acids and docosahexaenoic acid (DHA), an EFA abundant in fish. Calanoids and cladocerans contained similar levels of eicosapentaenoic acid (EPA), but cladocerans showed EPA : DHA ratios consistently greater than those of the available seston food source. Alpha-linolenic acid was most abundant in the herbivorous cladocerans, Daphnia and Holopedium , while the highest concentrations of arachidonic acid were found in the predatory cladocerans, Bythotrephes longimanus and Leptodora kindtii .
3. The distinct EFA accumulation patterns between cladoceran and copepod zooplankton suggest metabolic regulation of certain EFAs to meet the particular physiological demands and ecological strategies of these different zooplankton groups. Cladocerans may accumulate EPA directly from their diet, or through transformation of dietary materials to facilitate rapid somatic growth and enhance reproduction due to their short generation time. In contrast, copepods may retain DHA to increase their cell membrane fluidity in order to remain active over the winter due to their longer generation time and life cycle.
4. Consistent EFA differences between zooplankton groups may have implications regarding the somatic growth and reproductive success of different zooplankton taxa as well as the nutritional value of various zooplankton groups for larval and planktivorous fish.     

Modeling of eicosapentaenoic acid (EPA) production from Phaeodactylum tricornutum cultures in tubular photobioreactors. Effects of dilution rate, tube diameter, and solar irradiance

A model for the prediction of eicosapentaenoic acid (EPA) productivity from Phaeodactylum tricornutum cultures that takes into account the existence of photolimitation and photoinhibition of growth under outdoor conditions is presented. The effects of the external irradiance on the culture surface, the average irradiance inside the culture, and the light regime at which the cells are exposed on pigments and EPA content are studied. The chlorophyll content decreases exponentially with the average irradiance, whereas the carotenoids content increases linearly with the external irradiance due to a higher extension of photoinhibition. A decrease in the fatty acid content of the biomass with irradiance on reactor surface is observed when photoinhibition becomes relevant. The average irradiance within the culture mainly influenced the fatty acid profile of the biomass. As the average irradiance becomes higher, percentages of saturated and monounsaturated fatty acids decrease, increasing the portion of EPA. By taking into account the different relationships among pigment and EPA content with the irradiance, the variation in EPA productivity over the year can be simulated as a function of average and external irradiance. For the two photobioreactors employed the maximum EPA productivity is attained in spring and fall (30 mg L(-1) day(-1) for tube diameter 0. 06 m and 50 mg L(-1) day(-1) for tube diameter 0.03 m). In winter, the biomass productivity is limited by low light availability although the EPA content is maximum. In summer, the biomass productivity is higher although the EPA content diminished by photoinhibition; the higher the dilution rate, the lower the minimum. Thus, the conditions that increase the biomass productivity and the polyunsaturated fatty acids content are in opposition, the optimum being reached by operating under photolimitation with high growth rates in order to produce a high proportion of polyunsaturated fatty acids.     

Use of ciliate and phytoplankton taxonomic composition for the estimation of eicosapentaenoic acid concentration in lakes

1. The polyunsaturated fatty acid eicosapentaenoic acid (EPA) plays an important role in aquatic food webs, in particular at the primary producer–consumer interface where keystone species such as daphnids may be constrained by its dietary availability. Such constraints and their seasonal and interannual changes may be detected by continuous measurements of EPA concentrations. However, such EPA measurements became common only during the last two decades, whereas long‐term data sets on plankton biomass are available for many well‐studied lakes. Here, we test whether it is possible to estimate EPA concentrations from abiotic variables (light and temperature) and the biomass of prey organisms (e.g. ciliates, diatoms and cryptophytes) that potentially provide EPA for consumers. 2. We used multiple linear regression to relate size‐ and taxonomically resolved plankton biomass data and measurements of temperature and light intensity to directly measured EPA concentrations in Lake Constance during a whole year. First, we tested the predictability of EPA concentrations from the biomass of EPA‐rich organisms (diatoms, cryptophytes and ciliates). Secondly, we included the variables mean temperature and mean light intensity over the sampling depth (0–20 m) and depth (0–8 and 8–20 m) as factors in our model to check for large‐scale seasonal‐ and depth‐dependent effects on EPA concentrations. In a third step, we included the deviations of light and temperature from mean values in our model to allow for their potential influence on the biochemical composition of plankton organisms. We used the Akaike Information Criterion to determine the best models. 3. All approaches supported our proposition that the biomasses of specific plankton groups are variables from which seston EPA concentrations can be derived. The importance of ciliates as an EPA source in the seston was emphasised by their high weight in our models, although ciliates are neglected in most studies that link fatty acids to seston taxonomic composition. The large‐scale seasonal variability of light intensity and its interaction with diatom biomass were significant predictors of EPA concentrations. The deviation of temperature from mean values, accounting for a depth‐dependent effect on EPA concentrations, and its interaction with ciliate biomass were also variables with high predictive power. 4. The best models from the first and second approaches were validated with measurements of EPA concentrations from another year (1997). The estimation with the best model including only biomass explained 80%, and the best model from the second approach including mean temperature and depth explained 87% of the variability in EPA concentrations in 1997. 5. We show that it is possible to predict EPA concentrations reliably from plankton biomass, while the inclusion of abiotic factors led to results that were only partly consistent with expectations from laboratory studies. Our approach of including biotic predictors should be transferable to other systems and allow checking for biochemical constraints on primary consumers.     

Mixotrophic growth of Phaeodactylum tricornutum on glycerol: growth rate and fatty acid profile

Mixotrophic growth of the eicosapentaenoic acid (EPA)producing diatom Phaeodactylum tricornutum UTEX640 was carried out in 1-L batch cultures under anexternal irradiance of 165 mol photons m^-2s^-1 by supplementing the inorganic culture mediumwith glycerol. The effect on the growth and the fattyacid profile was studied for different initialglycerol concentrations (0–0.1 M). The optimalglycerol concentration was 0.1 M.A lag phase was observed at high glycerolconcentrations. The present study also shows thatsuccessive additions of glycerol at 0.1M concentrationand using ammonium chloride as a nitrogen sourceremarkably increased the maximum biomass concentration(16.2 g L^-1) and maximum biomass productivity(61.5 mg L^-1 h^-1). These values wererespectively 9 and 8-fold higher than in thephotoautotrophically grown control. The level ofsaponifiable lipids in mixotrophically cultured cellswas significantly higher than in photoautotrophicallycultured cells and increased with the glycerolconcentration in the medium. The concentration ofstorage lipids, saturated and monounsaturated fattyacids, were enhanced but the EPA content did notchange significantly. The EPA content was around 2.2%of biomass dry weight. The maximum EPA yield was33.5 mg L^-1 d^-1 and was obtained in aculture containing 0.1 M glycerol, supplementedperiodically by ammonium chloride. This productivitywas 10-fold higher than the EPA productivity obtainedunder mixotrophic conditions.     

Inhibitory effect of conjugated eicosapentaenoic acid on human DNA topoisomerases I and II

DNA topoisomerases (topos) and DNA polymerases (pols) are involved in many aspects of DNA metabolism such as replication reactions. We reported previously that long chain unsaturated fatty acids such as polyunsaturated fatty acids (PUFA) (i.e., eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA)) inhibited the activities of eukaryotic pols in vitro. In the present study, we found that PUFA also inhibited human topos I and II activities, and the inhibitory effect of conjugated fatty acids converted from EPA and DHA (cEPA and cDHA) on pols and topos was stronger than that of normal EPA and DHA. cEPA and cDHA inhibited the activities of mammalian pols and human topos, but did not affect the activities of plant and prokaryotic pols or other DNA metabolic enzymes tested. cEPA was a stronger inhibitor than cDHA with IC(50) values for mammalian pols and human topos of 11.0-31.8 and 0.5-2.5 microM, respectively. Therefore, the inhibitory effect of cEPA on topos was stronger than that on pols. Preincubation analysis suggested that cEPA directly bound both topos I and II, but did not bind or interact with substrate DNA. This is the first report that conjugated PUFA such as cEPA act as inhibitors of pols and topos. The results support the therapeutic potential of cEPA as a leading anti-cancer compound that poisons pols and topos.     

Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition

Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca²⁺-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the ω-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca²⁺-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca²⁺-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca²⁺ retention capacity and decreased Ca²⁺-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca²⁺-induced MPTP opening.     

Effect of oilseed type on milk fatty acid composition of individual cows,and also bulk tank milk fatty acid composition from commercial farms

Supplementing dairy cow diets with oilseed preparations has been shown to replace milk saturated fatty acids (SFA) with mono- and/or polyunsaturated fatty acids (MUFA, PUFA), which may reduce risk factors associated with cardio-metabolic diseases in humans consuming milk and dairy products. Previous studies demonstrating this are largely detailed, highly controlled experiments involving small numbers of animals, but in order to transfer this feeding strategy to commercial situations further studies are required involving whole herds varying in management practices. In experiment 1, three oilseed supplements (extruded linseed (EL), calcium salts of palm and linseed oil (CPLO) and milled rapeseed (MR)) were included in grass silage-based diets formulated to provide cows with ~350 g oil/day, and compared with a negative control (Control) diet containing no supplemental fat, and a positive control diet containing 350 g/cow per day oil as calcium salt of palm oil distillate (CPO). Diets were fed for 28-day periods in a 5×4 Latin Square design, and milk production, composition and fatty acid (FA) profile were analysed at the end of each period. Compared with Control, all lipid supplemented diets decreased milk fat SFA concentration by an average of 3.5 g/100 g FA, by replacement with both cis- and trans-MUFA/PUFA. Compared with CPO, only CPLO and MR resulted in lower milk SFA concentrations. In experiment 2, 24 commercial dairy farms (average herd size±SEM 191±19.3) from the south west of the United Kingdom were recruited and for a 1 month period asked to supplement their herd diets with either CPO, EL, CPLO or MR at the same inclusion level as the first study. Bulk tank milk was analysed weekly to determine FA concentration by Fourier Transform mid-IR spectroscopy prediction. After 4 weeks, EL, CPLO and MR all decreased herd milk SFA and increased MUFA to a similar extent (average −3.4 and +2.4 g/100 g FA, respectively) when compared with CPO. Differing responses observed between experiments 1 and 2 may be due in part to variations in farm management conditions (including basal diet) in experiment 2. This study demonstrates the importance of applying experimental research into commercial practice where variations in background conditions can augment different effects to those obtained under controlled conditions.     

Effect of exogenous sterols on the growth and fatty acid composition of the oomycetePythium debaryanum

Exogenous ergosterol and cholesterol were found to affect the growth and lipogenesis of the oomycete fungusPythium debaryanum, which is unable to synthesize de novo steroid compounds. These sterols stimulated the growth of the fungus during its submerged cultivation in glucose-peptone medium. This was accompanied by the shortening of the lag phase, the lengthening of the period of active growth, and by a 3.7-or 4.3-fold increase in the maximum biomass in response to the addition of ergosterol or cholesterol, respectively. In the presence of ergosterol, the cellular content of polyenoic fatty acids increased, and the relative content of eicosapolyenoic fatty acids reached 31.4% of the total amount of fatty acids in cells. Conversely, cholesterol decreased the cellular content of polyenoic acids, and the relative content of eicosapolyenoic acids fell to 19.6% of the total amount of fatty acids. It may be inferred that exogenous sterols enhance the yield of pharmacologically active polyenoic acids because of the growth stimulation.