Scale-Up to Pilot of a Non-Axenic Culture of Thraustochytrids Using Digestate from Methanization as Nitrogen Source.

The production of non-fish based docosahexaenoic acid (DHA) for feed and food has become a critical need in our global context of over-fishing. The industrial-scale production of DHA-rich Thraustochytrids could be an alternative, if costs turned out to be competitive. In order to reduce production costs, this study addresses the feasibility of the non-axenic (non-sterile) cultivation of Aurantiochytrium mangrovei on industrial substrates (as nitrogen and mineral sources and glucose syrup as carbon and energy sources), and its scale-up from laboratory (250 mL) to 500 L cultures. Pilot-scale reactors were airlift cylinders. Batch and fed-batch cultures were tested. Cultures over 38 to 62 h achieved a dry cell weight productivity of 3.3 to 5.5 g.L-1.day-1, and a substrate to biomass yield of up to 0.3. DHA productivity ranged from 10 to 0.18 mg.L-1.day-1. Biomass productivity appears linearly related to oxygen transfer rate. Bacterial contamination of cultures was low enough to avoid impacts on fatty acid composition of the biomass. A specific work on microbial risks assessment (in supplementary files) showed that the biomass can be securely used as feed. However, to date, there is a law void in EU legislation regarding the recycling of nitrogen from digestate from animal waste for microalgae biomass and its usage in animal feed. Overall, the proposed process appears similar to the industrial yeast production process (non-axenic heterotrophic process, dissolved oxygen supply limiting growth, similar cell size). Such similarity could help in further industrial developments.

Changes in lipid and fatty acid contents of gonad during the reproductive cycle of the Mediterranean swordfish Xiphias gladius.

Swordfish Xiphias gladius is a large pelagic fish distributed worldwide and exploited for human consumption, however there is limited knowledge about its reproductive biology, especially regarding lipid dynamic in gonads. In teleost fish, reproductive success and offspring survival are associated to lipid availability for gamete synthesis. This study investigated the lipid composition, including lipid classes and fatty acids (FA) of cell membrane and reserve lipids (i.e., polar and neutral lipids, respectively; PL and NL), along female and male gonad development of a swordfish population from waters surrounding Corsica Island in the Mediterranean Sea. Overall, swordfish gonads contained <2% wet weight of total lipids, with testes and ovaries having similar fat content. Lipid classes and FA concentrations remained unchanged during testes maturation. However, concentrations of phosphatidyl choline (PL), triacylglycerol (NL), and some FA (16:0, 18:1n-9, and 22:6n-3) followed an "inverted U-shaped" relationship with the ovarian maturation. In both PL and NL, 22:6n-3 was the main polyunsaturated FA (>20% of total FA), while 20:5n-3 and 20:4n-6 were minor (3-6% of total FA) and varied little with maturation. 22:6n-3 and 18:1n-9 were selectively allocated to the ovarian maturation (increased in concentration and in proportion with maturation) until spawning. Finally, swordfish gonads might represent a good food source for humans given that 150 g of swordfish ovaries can cover the daily requirements in omega-3 for humans, but research on pollutants should also be conducted to evaluate their implications on the reproduction output of this species, and on the safety of swordfish gonads for human consumption.

Risk and benefit assessment of seafood consumption harvested from the Pertuis Charentais region of France.

Seafood is well recognized as a major source of Long Chain n-3 Polyunsaturated Fatty Acids (LC n-3 PUFA, especially ecosapentaenoic acid, i.e. EPA and docosaheaxaenoic acid, i.e. DHA) and essential trace elements (As, Cu, Fe, Mn, Se, and Zn). It is also a source of non-essential trace elements (Ag, Cd, Hg, Pb) that can be deleterious for health even at low concentrations. Edible parts of sixteen species (fish, cephalopods, crustaceans and bivalves) of great importance in the Pertuis Charentais region, one of the main shellfish farming and fishing areas along the french coastline, were sampled in winter and analyzed to determine their fatty acid (FA) composition and trace element concentrations. Based on these analyses, a suite of indices was calculated to estimate risk and benefit of seafood consumption: the n-6/n-3 ratio, the atherogenic index, the thrombogenic index, the EPA + DHA daily recommended portion, as well as the maximum safe consumption. The results showed that fish contributed the most to LC n-3 PUFA supply, while bivalves and crustaceans were more beneficial in essential trace elements. Whatever the species, the concentrations of non-essential elements were not limiting for seafood consumption, as important amounts of the analyzed species can be eaten daily or weekly before becoming hazardous to consumers. Yet, concentrations of Hg in dogfish and seabass can become a concern for frequent seafood consumers (>three meals a week), confirming that varying seafood items is a key point for consumers to optimize the benefits of diverse seafood resources. Considering FA composition, whiting and pilchard are the most beneficial fish species for human diet, while surmullet was the least beneficial one. However, using an index integrating the relative risk due to Hg content, the surmullet appears as one of the most beneficial. This study provides a temporal shot of the quality of marine resources consumed in winter period in the studied area and highlights the complexity of a quantitative risk and benefit assessment with respect to the biochemical attributes of selected seafood.

Post-mortem storage conditions and cooking methods affect long-chain omega-3 fatty acid content in Atlantic mackerel (Scomber scombrus).

Long-chain omega-3 fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) are health beneficial lipids found in high concentration in pelagic fishes, including Atlantic mackerel. While EPA and DHA are sensitive to oxidation during fish storage and processing, post-mortem degradation in the first hours following fish death is poorly documented. Here, we stored fish at two temperatures (2-4 °C and 18-20 °C) and monitored EPA + DHA content in dorsal fillet 6, 12 and 24 h after fish death and after cooking (grill or steam). Storage duration was the only influencing factor, and EPA + DHA loss was faster at 18-20 °C. Six hours after fish death, EPA + DHA content decreased by 1.3 ± 1.3 mg.g-1 dw (9.6 ± 9.5% of the initial content) but it was highly variable among individuals. Handling between fishing and storage should be as short and as cool as possible to preserve EPA + DHA and food safety. Regarding cooking, EPA + DHA and mono-unsaturated fatty acids increased in grilled fillets.

A 13CO2 Enrichment Experiment to Study the Synthesis Pathways of Polyunsaturated Fatty Acids of the Haptophyte Tisochrysis lutea.

The production of polyunsaturated fatty acids (PUFA) in Tisochrysis lutea was studied using the gradual incorporation of a 13C-enriched isotopic marker, 13CO2, for 24 h during the exponential growth of the algae. The 13C enrichment of eleven fatty acids was followed to understand the synthetic pathways the most likely to form the essential polyunsaturated fatty acids 20:5n-3 (EPA) and 22:6n-3 (DHA) in T. lutea. The fatty acids 16:0, 18:1n-9 + 18:3n-3, 18:2n-6, and 22:5n-6 were the most enriched in 13C. On the contrary, 18:4n-3 and 18:5n-3 were the least enriched in 13C after long chain polyunsaturated fatty acids such as 20:5n-3 or 22:5n-3. The algae appeared to use different routes in parallel to form its polyunsaturated fatty acids. The use of the PKS pathway was hypothesized for polyunsaturated fatty acids with n-6 configuration (such as 22:5n-6) but might also exist for n-3 PUFA (especially 20:5n-3). With regard to the conventional n-3 PUFA pathway, Δ6 desaturation of 18:3n-3 appeared to be the most limiting step for T. lutea, "stopping" at the synthesis of 18:4n-3 and 18:5n-3. These two fatty acids were hypothesized to not undergo any further reaction of elongation and desaturation after being formed and were therefore considered "end-products". To circumvent this limiting synthetic route, Tisochrysis lutea seemed to have developed an alternative route via Δ8 desaturation to produce longer chain fatty acids such as 20:5n-3 and 22:5n-3. 22:6n-3 presented a lower enrichment and appeared to be produced by a combination of different pathways: the conventional n-3 PUFA pathway by desaturation of 22:5n-3, the alternative route of ω-3 desaturase using 22:5n-6 as precursor, and possibly the PKS pathway. In this study, PKS synthesis looked particularly effective for producing long chain polyunsaturated fatty acids. The rate of enrichment of these compounds hypothetically synthesized by PKS is remarkably fast, making undetectable the 13C incorporation into their precursors. Finally, we identified a protein cluster gathering PKS sequences of proteins that are hypothesized allowing n-3 PUFA synthesis.

Benefit-risk associated with the consumption of fish bycatch from tropical tuna fisheries.

Mercury, omega-3 (docosahexaenoic acid, DHA and eicosapentaenoic acid, EPA) and macronutrients (fat and proteins) were quantified on a wet weight (ww) basis in 20 species of fish taken as bycatch in tropical tuna fisheries. Based on a hazard quotient taking into account mercury and omega-3 contents, a benefit-risk assessment for the consumption of these pelagic species was conducted for three people categories: young children, children and adults. All fish bycatch were found to be an excellent source of proteins (min‒max = 14.4-25.2 g/100 g fillet), had low omega-6/omega-3 ratios (<1, except for silky shark), and had mercury content below the safety limits defined by sanitary agencies. Silky shark and Istiophoridae had the highest mercury contents (min‒max = 0.029-0.317 ppm ww). Omega-3 contents were the lowest in silky shark (0.2 ± 0.2 mg/100 g fillet) and the highest in striped marlin (3.6 ± 3.2 g/100 g fillet). Billfishes (Istiophoridae, including striped marlin), minor tunas (Scombridae), and Carangidae had the highest omega-3 contents (min‒max = 0.68-7.28 g/100 g fillet). The highest hazard quotient values obtained for silky shark and great barracuda reflected a lower nutritional benefit (i.e., low omega-3 source) than risk (i.e., mercury exposure), making them not advisable for consumption. Eight species had low hazard quotients, and among them cottonmouth jack and flat needlefish were found of high health interest (high protein, moderate fat contents, and low omega-6/omega-3 ratio). A daily serving portion of 85-200 g (according to people category) can be recommended for these species. Batfish, and to a lower extent pompano dolphinfish and brassy chub, can also be consumed safely and would provide greater health benefits than risks. These results advocate for a better access of these species to local populations.

Identification of Polyunsaturated Fatty Acids Synthesis Pathways in the Toxic Dinophyte Alexandrium minutum Using 13C-Labelling.

The synthetic pathways responsible for the production of the polyunsaturated fatty acids 22:6n-3 and 20:5n-3 were studied in the Dinophyte Alexandrium minutum. The purpose of this work was to follow the progressive incorporation of an isotopic label (13CO2) into 11 fatty acids to better understand the fatty acid synthesis pathways in A. minutum. The Dinophyte growth was monitored for 54 h using high-frequency sampling. A. minutum presented a growth in two phases. A lag phase was observed during the first 30 h of development and had been associated with the probable temporary encystment of Dinophyte cells. An exponential growth phase was then observed after t30. A. minutum rapidly incorporated 13C into 22:6n-3, which ended up being the most 13C-enriched polyunsaturated fatty acid (PUFA) in this experiment, with a higher 13C atomic enrichment than 18:4n-3, 18:5n-3, 20:5n-3, and 22:5n-3. Overall, the 13C atomic enrichment (AE) was inversely proportional to number of carbons in n-3 PUFA. C18 PUFAs, 18:4n-3, and 18:5n-3, were indeed among the least 13C-enriched FAs during this experiment. They were assumed to be produced by the n-3 PUFA pathway. However, they could not be further elongated or desaturated to produce n-3 C20-C22 PUFA, because the AEs of the n-3 C18 PUFAs were lower than those of the n-3 C20-C22 PUFAs. Thus, the especially high atomic enrichment of 22:6n-3 (55.8% and 54.9% in neutral lipids (NLs) and polar lipids (PLs), respectively) led us to hypothesize that this major PUFA was synthesized by an O2-independent Polyketide Synthase (PKS) pathway. Another parallel PKS, independent of the one leading to 22:6n-3, was also supposed to produce 20:5n-3. The inverse order of the 13C atomic enrichment for n-3 PUFAs was also suspected to be related to the possible ß-oxidation of long-chain n-3 PUFAs occurring during A. minutum encystment.

Study of Synthesis Pathways of the Essential Polyunsaturated Fatty Acid 20:5n-3 in the Diatom Chaetoceros Muelleri Using 13C-Isotope Labeling.

: The present study sought to characterize the synthesis pathways producing the essential polyunsaturated fatty acid (PUFA) 20:5n-3 (EPA). For this, the incorporation of 13C was experimentally monitored into 10 fatty acids (FA) during the growth of the diatom Chaetoceros muelleri for 24 h.Chaetoceros muelleri preferentially and quickly incorporated 13C into C18 PUFAs such as 18:2n-6 and 18:3n-6 as well as 16:0 and 16:1n-7, which were thus highly 13C-enriched. During the experiment, 20:5n-3 and 16:3n-4 were among the least-enriched fatty acids. The calculation of the enrichment percentage ratio of a fatty acid B over its suspected precursor A allowed us to suggest that the diatom produced 20:5n-3 (EPA) by a combination between the n-3 (via 18:4n-3) and n-6 (via 18:3n-6 and 20:4n-6) synthesis pathways as well as the alternative ω-3 desaturase pathway (via 20:4n-6). In addition, as FA from polar lipids were generally more enriched in 13C than FA from neutral lipids, particularly for 18:1n-9, 18:2n-6 and 18:3n-6, the existence of acyl-editing mechanisms and connectivity between polar and neutral lipid fatty acid pools were also hypothesized. Because 16:3n-4 and 20:5n-3 presented the same concentration and enrichment dynamics, a structural and metabolic link was proposed between these two PUFAs in C. muelleri.

Nanoplastics exposure modulate lipid and pigment compositions in diatoms.

The impact of nanoplastics (NP) using model polystyrene nanoparticles amine functionalized (PS-NH2) has been investigated on pigment and lipid compositions of the marine diatom Chaetoceros neogracile, at two growth phases using a low (0.05 µg mL-1) and a high (5 µg mL-1) concentrations for 96 h. Results evidenced an impact on pigment composition associated to the light-harvesting function and photoprotection mainly at exponential phase. NP also impacted lipid composition of diatoms with a re-adjustment of lipid classes and fatty acids noteworthy. Main changes upon NP exposure were observed in galactolipids and triacylglycerol's at both growth phases affecting the thylakoids membrane structure and cellular energy reserve of diatoms. Particularly, exponential cultures exposed to high NP concentration showed an impairment of long chain fatty acids synthesis. Changes in pigment and lipid content of diatom' cells revealed that algae physiology is determinant in the way cells adjust their thylakoid membrane composition to cope with NP contamination stress. Compositions of reserve and membrane lipids are proposed as sensitive markers to assess the impact of NP exposure, including at potential predicted environmental doses, on marine organisms.

An Integrated Biomarker Approach Using Flounder to Improve Chemical Risk Assessments in the Heavily Polluted Seine Estuary.

The objective of this study was to develop an integrative approach in ecotoxicology (from biomarkers to population genetics) to assess the ecological status of fish populations. Flounders (Platichthys flesus) collected after the spawning season in the heavily polluted Seine estuary were compared with the moderately polluted Bay of Douarnenez. The muscle energetic reserves were highly depleted in Seine vs. Douarnenez fish. The Seine fish displaying a reduced capacity to manage the oxidative stress and a higher energetic metabolism. An increase in the content of muscle membrane phospholipids (sphingomyelin, phosphatidylserine, free sterols) was detected in the Seine vs. Douarnenez fish. The data integration allowed to hypothesize relationships between membrane phospholipids, xenobiotic metabolism, bioenergetics, and antioxidant defence. The genetic diversity considering neutral markers was maintained in the heavily polluted Seine population compared with the Douarnenez population. Finally, we suggest that the high physiological cost of tolerance to toxicants in the Seine flounder population could compromise its capacity to respond in the future to an additional stressor like warming waters in shallow depth. Thus, this population could be submitted to an ecological risk.

Trophic ecology of suspension-feeding bivalves inhabiting a north-eastern Pacific coastal lagoon: Comparison of different biomarkers.

Understanding the nature and origins of food sources supporting coastal lagoon-inhabiting organisms is necessary to evaluate the ecological status of such ecosystems. The trophic ecology of a bivalve species Spondylus crassisquama was studied in the Ojo de Liebre lagoon (Baja California, Mexico), combining stable isotope (SI), fatty acid (FA) and sterol analyses along a transect under oceanic influences. The second objective of the study was to investigate if sterol compositions give complementary information to those obtained from FA and SI. Temporal and spatial patterns of the three biomarkers in bivalve tissues suggest oceanic inputs at the mouth of the lagoon, while the inner station was characterized by a contribution of local sources including an important role of micro heterotrophs. This study revealed that the association of lipid biomarkers provide higher taxonomic resolution of the spatiotemporal heterogeneity in nutrient sources influencing the trophic functioning of a large coastal lagoon.

Effects of extraction method and storage of dry tissue on marine lipids and fatty acids.

Various protocols are currently used to study marine lipids, but there is a growing interest in working on dry samples that are easier to transport. However, reference protocols are still lacking for dry samples. In order to make recommendations on this use, lipid classes and fatty acids (FA) obtained from six analytical protocols using two different tissue states (dry vs wet) and three extraction methods (automat vs manual potter vs leaving the solvent to work on tissue) were compared. Three dry storage modes of tissue (freezer vs gas nitrogen vs dry room) during one and three months were also compared. These comparisons were made on seven marine species with different lipid profiles, including fishes, crustaceans and mollusks. Lipid classes and FA obtained from wet and dry tissues were similar, but they were affected by the extraction methods. Regardless of tissue state, "Leave to work" methods obtained the highest lipid quantities, followed by manual potter and automat methods (ca. 90% and 80% of "Leave to work" methods, respectively). Linear relationships allowed correction for lipid classes and FA concentrations obtained from different protocols. The repeatability of all protocols still needs to be improved, especially for fish species. Increasing the replicate number for each sample might be an indirect way to improve lipid quantification. Our results show that storing dry tissues in the freezer for more than one month was associated with a decrease in lipids, which is also observed for other storage methods. For qualitative studies of FA (expressed in %), a three-month storage of dry tissue in freezer did not affect the relative composition of species/tissues with a lipid content below 20% of dry weight.