分离自象山港的15种海洋微藻脂肪酸比较研究

在160μmol.m-2.s-1光强、（20±2）℃条件下对分离自象山港的15种海洋微藻进行培养,在稳定期离心收集,冷冻干燥后用Bligh-Dyer法提取总脂,皂化衍生化后用气相色谱-质谱联用分析系统对其含有的脂肪酸进行定量和定性的分析。结果表明,这15种微藻总脂含量均较高,其中有9种微藻的总脂含量超过干重的10%,中心硅藻纲中共有的含量相对较高的主要脂肪酸为C14∶0、C16∶0、C16∶1（n-7）脂肪酸和EPA,针胞藻纲的赤潮异湾藻则含有高比例的C16∶0、C18∶4（n-3）和EPA,纵裂甲藻纲和甲藻纲中共有的含量相对较高的主要脂肪酸为C16∶0、C18∶4（n-3）、C18∶5（n-3）和DHA,而隶属于定鞭藻门颗石藻纲的颗石藻含量相对较高的主要脂肪酸分别为C16∶0、C18∶4（n-3）和DHA。这些藻是否可以作为生物饵料还需实际养殖投喂效果决定。     

Application of a growth-promoting bacteria for stable mass culture of three marine microalgae

The practical mass culture of marine microalgae, facesoccasionally unexpected problems or collapse. The effect of amarine bacterium, Flavobacterium sp., which was found topromote growth of a marine diatom Chaetoceros gracilisin the axenic culture condition, was examined on the masscultures of three marine microalgae.Three marine microalgae (C. gracilis, Isochrysisgalbana, and Pavlova lutheri) were mass cultured in 3 lflatbottom flasks (2.5 l capacity of culture medium), in anindoor culture room at a commercial pearl oyster hatchery. Themicroalgal cells and the bacterium were inoculated at the sametime, in the culture media. The specific growth rate andmaximal cell density were determined in treated cultures (withadded bacterial strain) and in controls (without addedbacterial strain). The specific growth rate of C.gracilis in treated cultures was significantly higher thanthat of control cultures, and the stationary growth phase inthe treated cultures lasted longer till the end of the cultureperiod. However, the bacterium had no apparent effect on theexponential growth phase of two phytoflagellates, I.galbana and P. lutheri, but kept longer the high celldensity in the stationary growth phases. The added bacterialstrain (Flavobacterium sp.) was the dominant species(more than 45%) among the bacterial flora during the cultureperiod.     

Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes; effects of n-3 and n-9 fatty acids

Atlantic salmon (Salmo salar) preadipocytes, isolated from visceral adipose tissue, differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. The expression of the adipogenic gene markers peroxisome proliferated activated receptor (PPAR) alpha, lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during differentiation. In addition, we describe a novel alternatively spliced form of PPARgamma (PPARgamma short), the expression of which increased during differentiation. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) lowered the triacylglycerol (TAG) accumulation in mature salmon adipocytes compared to oleic acid (18:1n-9, OA). This finding indicates that a reduced level of highly unsaturated n-3 fatty acids (HUFAs) in fish diets, when the traditional marine oil is exchanged for n-9 fatty acids (FAs) rich vegetable oils (VOs), may influence visceral fat deposition in salmonids. Moreover, major differences in the metabolism of EPA, DHA and OA at different stages during differentiation of adipocytes occur. Most of the EPA and DHA were oxidized in preadipocytes, while they were mainly stored in TAGs in mature adipocytes in contrast to OA which was primarily stored in TAGs at all stages of differentiation.     

Lipid compositions in diatom Conticribra weissflogii under static and aerated culture conditions

The diatom Conticribra weissflogii is a microalga with high nutrition value, rich in docosahexaenoic acids (DHA) and eicosapentaenoic acids (EPA). In order to study the effect of culture conditions on the changes of lipid compositions, the intact lipid structural profiles and fatty acids in C. weissflogii were monitored under static and aerated culture conditions. The results showed that, lipids identified in C. weissflogii were neutral lipid triacylglycerols (TAG), betaine lipid diacylglycerylcarboxy‐N‐hydroxymethyl‐choline (DGCC), phosphatidylcholine (PC) and four classes of photosynthetic glycerolipids. The profiles of lipid metabolites of C. weissflogii were different between two culture modes, with the following characteristics under aerated conditions: TAGs increased significantly, whereas the levels of sulfoquinovosyl diacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), and DGCC decreased. Furthermore, higher contents of EPA‐rich TAG and EPA/DHA‐rich DGCC were detected at the end of stationary phase, while EPA/DHA‐rich PC, EPA‐rich MGDG and EPA‐rich digalactosyldiacylglycerol (DGDG) were obtained in the exponential phase under static conditions. Meanwhile, the contents of almost all classes of the essential fatty acids (EFAs)‐enriched lipids increased at onset of stationary phase under aerated conditions. Taken together, given that the high levels of EFAs are required for artificial rearing of marine organisms, aeration is critically important for increasing the production rate and the contents of EFA molecules and therefore increasing the nutritional value of the microalgae.     

Increase of the yields of eicosapentaenoic and docosahexaenoic acids by the microalga Pavlova lutheri following random mutagenesis

The high commercial values of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids have driven a strain-improvement program, aimed at increasing the content of those fatty acids in the microalga Pavlova lutheri (SMBA 60) as parent strain. After a round of mutation using UV-light as mutagenic agent, an isolate strain (tentatively called II#2) was obtained, the EPA and DHA contents of which (in % dry biomass) were 32.8% and 32.9% higher than those of the control, native strain. The final EPA yields, when the cultures were maintained under appropriate conditions, were 17.4 and 23.1 mg. g(-1) dry biomass, for the wild-type and the II#2 strain, respectively, whereas the final DHA yields were 8.0 and 10.6 mg. g(-1) dry biomass, respectively. These results suggest that random mutagenesis can successfully be applied to increase the yield of n-3 fatty acids by microalgae.     

Production of BMAA and DAB by diatoms (Phaeodactylum tricornutum,Chaetoceros sp., Chaetoceros calcitrans and,Thalassiosira pseudonana) and bacteria isolated from a diatom culture

Microalgae have previously been reported to contain β-N-methylamino-l-alanine (BMAA), and the global presence of these primary producers has been associated with the widespread occurrence of BMAA in marine organisms. It has been repeatedly shown that filter-feeding bivalves accumulate phytoplankton species and their toxins. In this study, the concentrations of total soluble BMAA and DAB as a function of growth phase were observed for four non-axenic diatom species (i.e. Phaeodactylum tricornutum, Chaetoceros sp., Chaetoceros calcitrans and Thalassiosira pseudonana). These strains had previously been shown to contain BMAA using a highly selective HILIC-MS/MS method. BMAA cell quota appeared to be species-specific, however, highest BMAA concentrations were always obtained during the stationary growth phase, for all four species, suggesting that BMAA is a secondary metabolite. While DAB was detected in a bacterial culture isolated from a culture of P. tricornutum, the presence or absence of a bacterial population did not influence production of BMAA and DAB by P. tricornutum, i.e. no significant difference was noted for BMAA and DAB production between axenic and non-axenic cultures. The presence of DAB in bacteria had previously been shown, and raised the question as to whether DAB observed in many species of microalgae may arise from the non-axenic culture conditions or from the microalgae themselves.     

Isolation and Characterisation of a Δ5-fatty Acid Elongase from the Marine Microalga Pavlova salina

The marine microalga Pavlova salina (Haptophyta, Pavlovophyceae) produces lipids containing approximately 50% n-3 long-chain polyunsaturated fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). A full-length cDNA sequence, designated PsElo5, was isolated from P. salina. Sequence alignment showed that the gene was homologous to corresponding ELO-type elongases from other microalgae. Heterologous expression of PsElo5 in yeast and in higher plants confirmed that it encodes a specific Δ5-elongase activity as predicted and, furthermore, within the n-3 pathway, the elongation activity was confined exclusively to EPA.     

Identification of a very long chain polyunsaturated fatty acid Delta4-desaturase from the microalga Pavlova lutheri

Pavlova lutheri, a marine microalga, is rich in the very long chain polyunsaturated fatty acids (VLCPUFAs) eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids. Using an expressed sequence tag approach, we isolated a cDNA designated Pldes1, and encoding an amino acid sequence showing high similarity with polyunsaturated fatty acid front-end desaturases. Heterologous expression in yeast demonstrated that PlDES1 desaturated 22:5n-3 and 22:4n-6 into 22:6n-3 and 22:5n-6 respectively, and was equally active on both substrates. Thus, PlDES1 is a novel VLCPUFA Delta4-desaturase. Pldes1 expression is four-fold higher during the mid-exponential phase of growth compared to late exponential and stationary phases.     

Eicosapentaenoic acid and docosahexaenoic acid effects on tumour mitochondrial metabolism, acyl CoA metabolism and cell proliferation

In order to investigate the effects of high-fat diets rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), Wistar rats bearing subcutaneous implants of the Walker 256 tumour were fed pelleted chow containing low DHA/EPA or high DHA/EPA. The presence of n-3 polyunsaturated fatty acids (PUFAs) led to a marked suppression (35-46%) of tumour growth over a 12 day period. Both the whole tumour homogenate and the Percoll-purified mitochondrial fraction presented significant changes in fatty acid composition. The levels of EPA increased in both n-3 dietary groups while the levels of DHA increased only in the high DHA/EPA group, in comparison with the control chow-fed group. The presence of n-3 PUFAs led to an increase in mitochondrial acyl CoA synthetase activity, but neither the cytoplasmic acyl CoA content nor the n-3 fatty acid composition of the cytoplasmic acyl CoAs was altered by the diet. The content of thiobarbituric acid-reactive substances (TBARS) was increased in the low DHA/EPA group but was unchanged in the high DHA/EPA group. In vitro studies with the Walker 256 cell line showed a 46% decrease in cell growth in the presence of either EPA or DHA which was accompanied by a large decrease in the measured mitochondrial membrane potential. The TBARS content was increased only in the EPA-exposed cells. Cell cycle analysis identified a decrease in G0-G1 phase cells and an increase in G2-M phase cells and apoptotic cells, for both EPA and DHA-exposed cells. The data show that the presence of n-3 PUFAs in the diet is able to significantly after the growth rate of the Walker 256 tumour. The involvement of changes in mitochondrial membrane composition and membrane potential have been indicated for both EPA and DHA, while changes in lipid peroxidation have been identified in the presence of EPA but not of DHA.     

The role of marine omega-3 (n-3) fatty acids in inflammatory processes, atherosclerosis and plaque stability

Atherosclerosis has an important inflammatory component and acute cardiovascular events can be initiated by inflammatory processes occurring in advanced plaques. Fatty acids influence inflammation through a variety of mechanisms; many of these are mediated by, or associated with, the fatty acid composition of cell membranes. Human inflammatory cells are typically rich in the n-6 fatty acid arachidonic acid, but the contents of arachidonic acid and of the marine n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can be altered through oral administration of EPA and DHA. Eicosanoids produced from arachidonic acid have roles in inflammation. EPA also gives rise to eicosanoids and these are usually biologically weak. EPA and DHA give rise to resolvins which are anti-inflammatory and inflammation resolving. EPA and DHA also affect production of peptide mediators of inflammation (adhesion molecules, cytokines, etc.). Thus, the fatty acid composition of human inflammatory cells influences their function; the contents of arachidonic acid, EPA and DHA appear to be especially important. The anti-inflammatory effects of marine n-3 polyunsaturated fatty acids (PUFAs) may contribute to their protective actions towards atherosclerosis and plaque rupture.     

Grouping Newly Isolated Docosahexaenoic Acid-Producing Thraustochytrids Based on Their Polyunsaturated Fatty Acid Profiles and Comparative Analysis of 18S rRNA Genes

Seven strains of marine microbes producing a significant amount of docosahexaenoic acid (DHA; C22:6, n-3) were screened from seawater collected in coastal areas of Japan and Fiji. They accumulate their respective intermediate fatty acids in addition to DHA. There are 5 kinds of polyunsaturated fatty acid (PUFA) profiles which can be described as (1) DHA/docosapentaenoic acid (DPA; C22:5, n-6), (2) DHA/DPA/eicosapentaenoic acid (EPA; C20:5, n-3), (3) DHA/EPA, (4) DHA/DPA/EPA/arachidonic acid (AA; C20:4, n-6), and (5) DHA/DPA/EPA/AA/docosatetraenoic acid (C22:4, n-6). These isolates are proved to be new thraustochytrids by their specific insertion sequences in the 18S rRNA genes. The phylogenetic tree constructed by molecular analysis of 18S rRNA genes from the isolates and typical thraustochytrids shows that strains with the same PUFA profile form each monophyletic cluster. These results suggest that the C20-22 PUFA profile may be applicable as an effective characteristic for grouping thraustochytrids.     

Three-Year Breeding Cycle of Rainbow Trout (Oncorhynchus mykiss) Fed a Plant-Based Diet,Totally Free of Marine Resources: Consequences for Reproduction,Fatty Acid Composition and Progeny Survival

Terrestrial plant resources are increasingly used as substitutes for fish meal and fish oil in fish feed in order to reduce the reliance of aquaculture on marine fishery resources. Although many studies have been conducted to assess the effects of such nutritional transition, no whole breeding cycles of fish fed diets free from marine resources has been reported to date. We therefore studied the reproductive performance of trout after a complete cycle of breeding while consuming a diet totally devoid of marine ingredients and thus of n-3 long chain polyunsaturated fatty acids (n-3 LC-PUFAs) that play a major role in the formation of ova. Two groups of female rainbow trout were fed from first feeding either a commercial diet (C, marine and plant ingredients), or a 100% plant-based diet (V, blend of plant proteins and vegetable oils). Livers, viscera, carcasses and ova were sampled at spawning and analyzed for lipids and fatty acids. Although the V-diet was devoid of n-3 LC-PUFAs, significant amounts of EPA and DHA were found in livers and ova, demonstrating efficient bioconversion of linolenic acid and selective orientation towards the ova. Some ova were fertilized to assess the reproductive performance and offspring survival. We observed for the first time that trout fed a 100% plant-based diet over a 3-year breeding cycle were able to produce ova and viable alevins, although the ova were smaller. The survival of offspring from V-fed females was lower (-22%) at first spawning, but not at the second. Our study showed that, in addition to being able to grow on a plant-based diet, rainbow trout reared entirely on such a diet can successfully produce ova in which neo-synthesized n-3 LC-PUFAs are accumulated, leading to viable offspring. However, further adjustment of the feed formula is still needed to optimize reproductive performance.     

Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production

ABSTRACT: Omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) provide significant health benefits and this has led to an increased consumption as dietary supplements. Omega-3 fatty acids EPA and DHA are found in animals, transgenic plants, fungi and many microorganisms but are typically extracted from fatty fish, putting additional pressures on global fish stocks. As primary producers, many marine microalgae are rich in EPA (C20:5) and DHA (C22:6) and present a promising source of omega-3 fatty acids. Several heterotrophic microalgae have been used as biofactories for omega-3 fatty acids commercially, but a strong interest in autotrophic microalgae has emerged in recent years as microalgae are being developed as biofuel crops. This paper provides an overview of microalgal biotechnology and production platforms for the development of omega-3 fatty acids EPA and DHA. It refers to implications in current biotechnological uses of microalgae as aquaculture feed and future biofuel crops and explores potential applications of metabolic engineering and selective breeding to accumulate large amounts of omega-3 fatty acids in autotrophic microalgae.     

Changes in the lipid composition and maximisation of the polyunsaturated fatty acid content of three microalgae grown in mass culture

Three species of microalgae were grown in mass culture to investigate the influence of culture technique and growth phase on the production of 20:5(n?3) and 22:6(n?3). These polyunsaturated fatty acids (PUFA) are considered to be essential in many marine animals diets for high growth and survival rates. The species of microalgae examined wereNannochloropsis oculata, Pavlova lutheri andIsochrysis sp. (clone T.Iso). All batch cultures (logarithmic and stationary phase) and semi-continuous cultures (logarithmic phase) examined contained high levels of the long-chain (n?3) PUFA, but production could be maximised by harvesting at specific times and growth phases. Maximum cellular content (pg cell^-1) of long-chain PUFA was found in logarithmic phase batch cultures ofN. oculata and in stationary phase cultures ofP. lutheri. The cellular content of PUFA in cultures ofIsochrysis sp. did not change significantly with culture technique or growth phase. Alternatively, stationary phase cultures of all three species showed increased proportions (%) and cellular contents of triacylglycerols, and saturated and monounsaturated fatty acids with correspondingly decreased proportions of polar lipids and most PUFA relative to logarithmic phase cultures. The exception was the proportion and cellular content of 22:6(n?3) inP. lutheri which increased with triacylglycerol content. The mass of long-chain (n?3) PUFA per volume of culture was significantly higher in stationary phase cultures due to the higher cell counts per volume. These findings indicate that the opportunity exists to maximise PUFA production by microalgae with the potential to improve animal growth and reduce production costs in mariculture operations and may be of use in the large scale culture and harvesting of microalgae for the biotechnology industry.     

Activation and deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in three marine microalgae

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was examined in three marine microalgae: the chlorophyte t Dunaliella tertiolecta and the chromophytes t Pavlova lutheri and t Thalassiosira pseudonana. The three species differed in the sensitivity of Rubisco activity in crude extracts to magnesium ion concentration, the presence of protease inhibitors, the duration of the incubation on activity, and the potential for full activation of Rubisco with 20 mM magnesium chloride and 20 mM bicarbonate t in vitro. t D. tertiolecta had responses that were similar to those described in vascular plants: regulation of initial activity on a gradient of irradiances; maximum initial activities that were 80– 90% of light-saturated photosynthesis; total activities that exceeded light-saturated photosynthesis by 30–100%; and deactivation of Rubisco in darkness. Both initial and total activity declined in darkness and increased on a return to growth irradiance. First-order time constants were about 9 min for deactivation and 3 min for reactivation of initial activity. The decline in total activity after a transition into darkness could not be reversed t in vitro but could be reversed by exposing t D. tertiolecta to light, a characteristic of regulation by CA1P. The responses of t T. pseudonana were qualitatively similar, except that recovery of initial activity was low and could only account for 30–40% of light-saturated photosynthesis. Rubisco from t T. pseudonana exposed to low irradiance could be activated t in vitro but at growth irradiance and higher, total activity was lower than initial activity. The time constants for deactivation and reactivation of initial activity after reciprocal switches between growth irradiance and darkness were 12–18 min and 3 min in t T. pseudonana. t P. lutheri showed no regulation of Rubisco activity in response to changes in irradiance or light-dark transitions. This may have been an artifact of the conditions chosen to measure activity.     

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。     

Triacylglycerol profiling of marine microalgae by mass spectrometry

We present a method for the determination of triacylglycerol (TAG) profiles of oleaginous saltwater microalgae relevant for the production of biofuels, bioactive lipids, and high-value lipid-based chemical precursors. We describe a technique to remove chlorophyll using quick, simple solid phase extraction (SPE) and directly compare the intact TAG composition of four microalgae species (Phaeodactylum tricornutum, Nannochloropsis salina, Nannochloropsis oculata, and Tetraselmis suecica) using MALDI time-of-flight (TOF) mass spectrometry (MS), ESI linear ion trap-orbitrap (LTQ Orbitrap) MS, and 1H NMR spectroscopy. Direct MS analysis is particularly effective to compare the polyunsaturated fatty acid (PUFA) composition for triacylglycerols because oxidation can often degrade samples upon derivatization. Using these methods, we observed that T. suecica contains significant PUFA levels with respect to other microalgae. This method is applicable for high-throughput MS screening of microalgae TAG profiles and may aid in the commercial development of biofuels.     

Comparative anti-inflammatory effects of plant- and marine-derived omega-3 fatty acids explored in an endothelial cell line

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) lower risk of cardiovascular disease. The primary source of EPA and DHA is fatty fish. Plant-derived alpha linolenic acid (ALA) and stearidonic acid (SDA) could provide sustainable land-based alternatives, but their functionality is underexplored. Omega-3 fatty acids (n-3 FAs) may influence atherogenic processes through changing endothelial cell (EC) function and lowering inflammation. This study compared effects of marine- and plant-derived n-3 FAs on EC inflammatory responses. EA.hy926 cells were exposed to ALA, SDA, EPA or DHA prior to stimulation with tumor necrosis factor (TNF)-α. All FAs were shown to be incorporated into ECs in a dose-dependent manner. SDA (50 μM) decreased both production and cell-surface expression of intercellular adhesion molecule (ICAM)-1; however EPA and DHA resulted in greater reduction of ICAM-1 production and expression. EPA and DHA also significantly lowered production of monocyte chemoattractant protein 1, interleukin (IL)-6 and IL-8. ALA, SDA and DHA (50 μM) all reduced adhesion of THP-1 monocytes to EA.hy926 cells. DHA significantly decreased nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)p105 gene expression and phosphorylated NFκBp65 protein. Both EPA and DHA (50 μM) significantly decreased cyclooxygenase (COX)-2 protein. Thus, both marine-derived n-3 FAs, particularly DHA, had potent anti-inflammatory effects in this EC model. Of the plant-derived n-3 FAs, SDA showed the greatest inhibition of inflammation. Although neither ALA nor SDA reproduced the anti-inflammatory effects of EPA and DHA in this model, there is some potential for SDA to be a sustainable anti-inflammatory alternative to the marine n-3 FAs.