Search

The newly recorded Phyllymenia gibesii in the Mediterranean Sea at Alexandria coast of Egypt is regarded as a significant source of bioactive substances and is applied as an antioxidant, anti-inflammatory, and antimicrobial agent. According to the HPLC chromatograms, the acetone extract of P. gibesii comprised ten photosynthetic pigments (chlorophyll-a, chlorophyll-d, α-carotene, β-carotene, phycocyanin, allophycocyanin, antheraxanthin, β-cryptoxanthin, lutein, and violaxanthin). Total carotenoids were the dominant class in the pigments' profile, achieving a concentration of 257 g/g dry weight. The P. gibbesii extract had a total content of phenols (146.67 mg/g) and a total content of flavonoids (104.40 mg/g). The capacity of all the investigated biological activities augmented with the concentration of the algal extract. The maximal DPPH scavenging capacity was 81.44%, with an inhibitory concentration (IC 50 ) of 9.88 μg/mL. Additionally, the highest ABTS scavenging capacity was 89.62%, recording an IC 50 of 21.77 μg/mL. The hemolytic activity of P. gibbesii attained a maximum capacity of 49.88% with an IC 50 of 100.25 μg/mL. Data also showed the maximum anti-inflammatory effectiveness at 81.25%, with an IC 50 of 99.75 μg/mL. Furthermore, the extract exhibited antimicrobial capacity against all reference strains, particularly at high concentrations (0.1 mg/mL), with the greatest effect on C. albicans and E. coli., (© 2024. The Author(s).)

Schizochytrium sp. (SZ) can potentially be employed in nutritional strategies for producing high-quality sheep meat. However, the effects of SZ on the lipid composition of sheep meat are insufficiently understood. In this study, the effects of SZ supplementation on the lipid profile of Tan sheep meat were evaluated using non-targeted lipidomic techniques. Lipidomics analysis revealed 383 differential lipids (DLs) between the SZ and control groups, and there were six metabolic pathways associated with lipids, including glycerophospholipid metabolism, glycerolipid metabolism, α-linolenic acid metabolism, linoleic acid metabolism, glycine, serine and threonine metabolism, and arachidonic acid metabolism (P < 0.05). Glycerophospholipid metabolism was the core pathway of DLs; we found that phosphatidylcholine, phosphatidylserine, and lysophosphatidylcholine were the crucial lipid metabolites of this pathway. Dietary supplementation with SZ increased n-3 polyunsaturated fatty acid (PUFA), C22:6n-3, and C20:5n-3 (P < 0.05), while it decreased C18:0, saturated fatty acid (SFA), and SFA/PUFA (P < 0.05). These results indicate that SZ supplementation induces positive alterations in the lipid profile of Tan sheep meat, which is beneficial to meat quality and sheds valuable insights into the future development of functional lipids in sheep meat., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The oil from the heterotroph Schizochytrium is a rich source of n-3 PUFA, particularly DHA, and therefore highly susceptible to oxidation. The present work reports the first application of coaxial prilling for the protection of this oil through microencapsulation. After process optimization, core-shell microparticles were produced with calcium or zinc alginate at different concentrations. Encapsulates were analyzed in their tocopherol and PUFA content. Prilling lowered the earlier but had little effect on the latter. Microcapsules coated with calcium alginate (1 % and 1.75 %) had higher oil load and encapsulation efficiency and were therefore submitted to in vitro digestion together with a simulated meal. Digesta were also analyzed with HPLC-qTOF and 1 H NMR and compared to undigested encapsulates. While 1 % calcium shell granted lower oil release and protection from oxidation in the simulated gastrointestinal tract, chromatographic and spectroscopic data of digesta showed higher presence of lipid digestion products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Climate change and global warming have led to more frequent harmful algal blooms in the last decade. Among these blooms, Heterosigma akashiwo, a golden-brown phytoflagellate, is one of the 40 species with a high potential to form harmful blooms, leading to significant fish mortality. Climate change leads to rising atmospheric and ocean temperatures. These changes, along with altered rainfall patterns and meltwater input, can cause fluctuations in ocean salinity. Elevated atmospheric carbon dioxide (CO 2 ) levels increase water acidity as oceans absorb CO 2 . This study investigated the effects of temperature, salinity, and CO 2 levels on lipid production, hemolytic activity, and toxicity of H. akashiwo using the design of experiment approach, which can be used to investigate the effect of two or more factors on the same response simultaneously in a precise manner with fewer experiments and materials but in a larger region of the factor space. The lipid content was measured using a high-throughput Nile Red method, and the highest level of lipid content was detected at 25°C, a salinity of 30, and a CO 2 concentration of 400 ppm. Hemolytic activity was assessed using rabbit blood erythrocytes in a 96-well plate, and the optimal conditions for achieving the highest hemolytic activity were determined at 15°C, a salinity of 10, and a CO 2 concentration of 400 ppm. As the chemical structure of the toxin is not known, we used the toxicity against the cell line RTgill-W1 as the cell toxicity proxy. The maximum toxicity was identified at 15°C, a salinity of 10, and a CO 2 level of 700 ppm., (© 2024 The Author(s). Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.)

Few reports exist on one-step enzymatic methods for the simultaneous production of biodiesel and eicosapentaenoic acid ethyl ester (EPA-EE), a high-value pharmaceutical compound. This study aimed to efficiently express Rhizomucor miehei lipase (pRML) in Pichia pastoris X-33 via propeptide mutation and high-copy strain screening. The mutated enzyme was then used to simultaneously catalyze the production of both biodiesel and EPA-EE. The P46N mutation in the propeptide (P46N-pRML) significantly boosted its production, with the four-copy strain increasing enzyme yield by 3.7-fold, reaching 3425 U/mL. Meanwhile, its optimal temperature increased to 45-50 °C, pH expanded to 7.0-8.0, specific activity doubled, K m reduced to one-third, and k cat / K m increased 7-fold. Notably, P46N-pRML efficiently converts Nannochloropsis gaditana oil's eicosapentaenoic acid (EPA). Under optimal conditions, it achieves up to 93% biodiesel and 92% EPA-EE yields in 9 h. Our study introduces a novel, efficient one-step green method to produce both biodiesel and EPA-EE using this advanced enzyme.

Microalgae, integral to marine ecosystems for their rich nutrient content, notably lipids and proteins, were investigated by using reversed-phase liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (RPLC-Q-TOF-MS/MS). This study focused on lipid composition in three commonly used microalgae species ( Spirulina platensis , Chlorella vulgaris , and Schizochytrium limacinum ) for functional food applications. The analysis unveiled more than 700 lipid molecular species, including glycolipids (GLs), phospholipids (PLs), sphingolipids (SLs), glycerolipids, and betaine lipids (BLs). GLs (19.9-64.8%) and glycerolipids (24.1-70.4%) comprised the primary lipid. Some novel lipid content, such as acylated monogalactosyldiacylglycerols (acMGDG) and acylated digalactosyldiacylglycerols (acDGDG), ranged from 0.62 to 9.68%. The analysis revealed substantial GLs, PLs, and glycerolipid variations across microalgae species. Notably, S. platensis and C. vulgaris displayed a predominance of fatty acid (FA) 18:2 and FA 18:3 in GLs, while S. limacinum exhibited a prevalence of FA 16:0, collectively constituting over 60% of the FAs of GLs. In terms of PLs and glycerolipids, S. platensis and C. vulgaris displayed elevated levels of arachidonic acid (AA) and eicosapentaenoic acid (EPA), whereas S. limacinum exhibited a significant presence of docosahexaenoic acid (DHA). Principal component analysis (PCA) revealed MGDG (16:0/18:1), DG (16:0/22:5), Cer (d18:1/20:0), and LPC (16:1) as promising lipid markers for discriminating between these microalgae samples. This study contributes to a comprehensive understanding of lipid profiles in three microalgae species, emphasizing their distinct biochemical characteristics and potentially informing us of their high-value utilization in the food industry.

This study examined the effects of high-pressure homogenization (HPH) and ultrasonication pre-treatment on the structural and physicochemical properties of proteins extracted from defatted Nannochloropsis Oceania biomass (DNOB). HPH treatment was found to enhance the solubility of protein extracted from DNOB compared to ultrasound, where samples pretreated with three passes (3P) of HPH exhibited lower solubility than two passes (2P). The morphology of extracted samples was visualized by scanning electron microscopy, which HPH pre-treatment, especially with more passes, were able to breakdown DNOB into fragments. Alternatively, more holes were displayed on the surface of the extracts pretreated with ultrasound especially when higher amplitude applied. The particle size of extracts from HPH 3P (129.5 µm) significant dropped from HPH 2P (314.25 µm), where samples pretreated with ultrasound at 20 % amplitude (US 20 ) also decreased in particle size compared to 40 % amplitude (US 40 ), from 115.25 µm to 78.22 µm. Protein flexibility of DNOB extracts were enhanced by both HPH 2P and HPH 3P but decreased for ultrasound samples. β-sheets were found to be the most abundant protein secondary structure for all samples, where samples treated with HPH 3P contained the highest percentage of β-sheets (72 %) than control, HPH 2P , ultrasonication at 20 and 40 % amplitude (52-62 %). The high percentage of β-sheets found in HPH 3P sample also contributed to its outstanding emulsifying properties which stood out among all, especially at concentrations over 1 mg/ml. Results obtained from this study helped to direct the application of DNOB extracts as functional food ingredient for future food innovation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Marine microalgae Schizochytrium sp. have a high content of docosahexaenoic acid (DHA), an omega-3 fatty acid that is attracting interest since it prevents certain neurodegenerative diseases. The obtention of a bioactive and purified DHA fatty acid ester using a whole-integrated process in which renewable sources and alternative methodologies are employed is the aim of this study. For this reason, lyophilized Schizochytrium biomass was used as an alternative to fish oil, and advanced extraction techniques as well as enzymatic modification were studied. Microalgal oil extraction was optimized via a surface-response method using pressurized liquid extraction (PLE) obtaining high oil yields (29.06 ± 0.12%) with a high concentration of DHA (51.15 ± 0.72%). Then, the enzymatic modification of Schizochytrium oil was developed by ethanolysis using immobilized Candida antarctica B lipase (Novozym ® 435) at two reaction temperatures and different enzymatic loads. The best condition (40 °C and 200 mg of lipase) produced the highest yield of fatty acid ethyl ester (FAEE) (100%) after 8 h of a reaction attaining a cost-effective and alternative process. Finally, an enriched and purified fraction containing DHA-FAEE was obtained using open-column chromatography with a remarkably high concentration of 93.2 ± 1.3% DHA. The purified and bioactive molecules obtained in this study can be used as nutraceutical and active pharmaceutical intermediates of marine origin.

Background: The rise of antibiotic-resistant bacteria necessitates the discovery of new, safe, and bioactive antimicrobial compounds. The antibacterial and antibiofilm activity of microalgae makes them a potential candidate for developing natural antibiotics to limit microbial infection in various fields., Objective: This study aimed to analyze the antibacterial effect of the methanolic extract of Schizochytrium sp. S31 microalgae by broth microdilution and spot plate assays., Methods: The antibacterial effects of Schizochytrium sp. S31 extract was studied on gramnegative pathogens, Pseudomonas aeruginosa, Escherichia coli 35218, Klebsiella pneumonia, which cause many different human infections, and the gram-positive pathogen Streptococcus mutans. At the same time, the antibiofilm activity of the Schizochytrium sp. S31 extract on Pseudomonas aeruginosa and Escherichia coli 35218 bacteria were investigated by crystal violet staining method., Results: Schizochytrium sp. S31 extract at a 60% concentration for 8 hours displayed the highest antibacterial activity against P. aeruginosa, E. coli 35218, and K. pneumonia, with a decrease of 87%, 92%, and 98% in cell viability, respectively. The experiment with Streptococcus mutans revealed a remarkable antibacterial effect at a 60% extract concentration for 24 hours, leading to a notable 93% reduction in cell viability. Furthermore, the extract exhibited a dose-dependent inhibition of biofilm formation in P. aeruginosa and E. coli 35218. The concentration of 60% extract was identified as the most effective dosage in terms of inhibition., Conclusion: This research emphasizes the potential of Schizochytrium sp. S31 as a natural antibacterial and antibiofilm agent with promising applications in the pharmaceutical sectors. This is the first study to examine the antibacterial activity of Schizochytrium sp. S31 microalgae using broth microdilution, spot plate assays, and the antibiofilm activity by a crystal staining method. The findings of this study show that Schizochytrium sp. S31 has antibacterial and antibiofilm activities against critical bacterial pathogens., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Microalgae offer an opportunity to act as a sustainable source of dietary protein. This study aimed to evaluate the impact of different protein extraction methods on the nutritional and physicochemical properties of Nannochloropsis oculata. Food-grade protein extracts were obtained by hypotonic osmotic shock using milli-Q water. Food grade (FG) and non-food grade (NFG) extraction buffers were compared along with three cell disruption methods including bead beating, probe sonication and a combination of both methods for protein extraction. Mass spectrometry was used for protein and putative allergen identification in FG extracts. Bead beating led to a slightly higher number of identifiable proteins in FG extracts compared to control condition. Putative allergenic proteins were identified in FG extracts of N. oculata using different in-silico methods. These findings support the need to further evaluate the potential allergenic proteins in microalgae including N. oculata such as immunoglobulin E (IgE) binding tests., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2023. Published by Elsevier Ltd. All rights reserved.)

Several species of microalgae are promising as an alternative source of omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). Photoautotrophic species show the greatest potential, since incorporating them into food products leads to oxidatively stable products; however, the presence of photosensitizers could reduce the shelf-life due to the appearance of photo-oxidation on exposure to light. This study investigated the oxidative impact of illumination for aqueous model suspensions enriched with Phaeodactylum (phototrophic microalgae─containing potential photosensitizers) and Schizochytrium (heterotrophic microalgae─lacking photosensitizers) during storage for 28 days at 37 °C. Primary (peroxide value) and secondary (volatiles with gas chromatography (GC)-mass spectrometry) oxidation products, n-3 LC-PUFA content (GC), and pigments (high-pressure liquid chromatography) were assessed. The results showed that photo-oxidation did not cause oxidative instability for Phaeodactylum samples compared with strong autoxidation in Schizochytrium samples. For the Phaeodactylum- enriched suspensions, only minimal photo-oxidation could be detected and the n-3 LC-PUFA content remained stable throughout storage regardless of illumination.

Harmful algal blooms formed by fast-growing, ephemeral macroalgae have expanded worldwide, yet there is limited knowledge of their potential ecological consequences. Here, we select intense green tides formed by Ulva prolifera in the Yellow Sea, China, to examine the ecological consequences of these blooms. Using 28-isofucosterol in the surface sediment as a biomarker of green algae, we identified the settlement region of massive floating green algae in the area southeast of the Shandong Peninsula in the southern Yellow Sea. The responses of the phytoplankton assemblage from the deep chlorophyll- a maximum layer were then resolved using high-throughput sequencing. We found striking changes in the phytoplankton community in the settlement region after an intensive green tide in 2016, characterized by a remarkable increase in the abundance of the pelagophyte Aureococcus anophagefferens , the causative species of ecosystem disruptive brown tides. Our study strongly suggests that the occurrence of massive macroalgal blooms may promote blooms of specific groups of microalgae through alteration of the marine environment.

Improved methods for the extraction of eicosapentaenoic acid (EPA), an essential and economically important polyunsaturated fatty acid, are urgently required. However, lipid extraction rates using food-grade solvents such as ethanol are usually low. To improve the ethanol-based extraction rate, and to elucidate the relevant mechanisms, we used cellulase and laccase to treat powdered Nannochloropsis , one of the most promising microalgal sources of EPA. Cellulase and laccase synergistically increased lipid yields by 69.31% and lipid EPA content by 42.63%, by degrading the amorphous hemicellulose and cellulose, improving crystallinity, and promoting the release and extraction of lysodiacylglyceryltrimethylhomoserine. Scanning electron microscopy showed that cell morphology was substantially altered, with cell-wall rupture, loss of cell boundaries, and the release of intracellular substances. In conclusion, Nannochloropsis lipid yields may be directly linked to cell-wall hemicellulose structure, and enzymatic treatment to alter this may improve lipid yields.

We investigated whether dietary supplementation with Aurantiochytrium sp. meal, a DHA-rich source (docosahexaenoic acid, 22: 6 n-3), fed during long-term exposure to cold-suboptimal temperature (22 °C, P1), followed by short-term exposure to higher temperatures (28 °C, P2, and 33 °C, P3), would promote oxidative damage in Nile tilapia (Oreochromis niloticus). Two supplementation levels were tested: 1.0 g 100 g -1 (D1) and 4.0 g 100 g -1 (D4). A control diet, without the additive (D0, 0 g 100 g -1 ), and a positive control diet supplemented with cod liver oil (CLO) were also tested. The concentrations of DHA and total n-3 PUFAs in the CLO diet were similar to those found in diets D1 and D4, respectively. The parameters analyzed included hemoglobin (Hb), the antioxidant enzymes catalase, glutathione peroxidase, total glutathione, non-protein thiols, and the oxidative markers protein carbonyl and erythrocyte DNA damage. Nile tilapia did not present differences in Hb content, regardless of diet composition, but the temperature increase (P1 to P2) led to a higher Hb content. Likewise, the temperature increases promoted alterations in all antioxidant enzymes. The dietary supplementation with 1.0 g 100 g -1 Aurantiochytrium sp. meal after P1 caused minor DNA damage in Nile tilapia, demonstrating that the additive can safely be included in winter diets, despite its high DHA concentration., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Polyunsaturated fatty acid (PUFA) synthase is a special and effective enzyme for PUFA synthesis, and dehydratase (DH) domain played a crucial role in it. In this work, we compared four different DH domains from different strains (Schizochytrium sp. HX-308 and Shewanella sp. BR-2) and different gene clusters. First bioinformatics analysis showed that DH 1, 2 and DH 3 were similar to FabA and PKS-DH, respectively, and all of them got a hot-dog structure. Second, four DH domains were expressed in Escherichia coli that increased biomass. Especially, Schi-DH 1,2 presented the highest dry cell weight of 2.3 g/L which was 1.62 times of that of control. Fatty acids profile analysis showed that DH 1,2 could enhance the percentage of unsaturated fatty acids, especially DH 1,2 from Schizochytrium sp., while DH 3 benefited for the saturated fatty acid biosynthesis. Furthermore, five kinds of fatty acids were added to the medium to study the substrate preferences. Results revealed that DH 1,2 domain preferred to acting on C16:0, while DH 3 domain trended acting on C14:0 and C15:0, which illustrated DH from different clusters do have specific substrate preference. Besides, DH expression could save the cell growth inhibition by mid-chain fatty acids. This study provided more information about the catalysis mechanism of polyunsaturated fatty acid synthase and might promote the modification study based on this enzyme., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Eicosapentaenoic acid (EPA) from freeze-dried biomass of Nannochloropsis oceanica microalgae resists ruminal biohydrogenation in vitro, but in vivo demonstration is needed. Therefore, the present study was designed to test the rumen protective effects of N. oceanica in lambs. Twenty-eight lambs were assigned to one of four diets: Control (C); and C diets supplemented with: 1.2% Nannochloropsis sp. oil (O); 12.3% spray-dried N. oceanica (SD); or 9.2% N. oceanica (FD), to achieve 3 g EPA /kg dry matter. Lambs were slaughtered after 3 weeks and digestive contents and ruminal wall samples were collected. EPA concentration in the rumen of lambs fed FD was about 50% higher than lambs fed SD or O diets. Nevertheless, the high levels of EPA in cecum and faeces of animals fed N. oceanica biomass, independently of the drying method, suggests that EPA was not completely released and absorbed in the small intestine. Furthermore, supplementation with EPA sources also affected the ruminal biohydrogenation of C18 fatty acids, mitigating the shift from the t10 biohydrogenation pathways to the t11 pathways compared to the Control diet. Overall, our results demonstrate that FD N. oceanica biomass is a natural rumen-protected source of EPA to ruminants., (© 2021. The Author(s).)

The element stoichiometry of bacteria has received considerable attention because of their significant role in marine ecosystems. However, relatively little is known about the composition of major structural elements of the unicellular heterotrophic protists-thraustochytrids, despite their widely recognized contribution to marine nutrient cycling. Here, we analyze the cell volume and elemental C, N, H, and S cell content of seven cultured thraustochytrids, isolated from different marine habitats, in the exponential and stationary growth phases. We further derive the relationships between the cell volume and elemental C and N content of the cultured thraustochytrids. The cell volumes varied significantly ( p < 0.001) among the isolates, with median values of 96.9 and 212.5 μm 3 in the exponential and stationary phases, respectively. Our results showed a significantly higher percentage of C (64.0 to 67.5) and H (9.9 to 13.2) but a lower percentage of N (1.86 to 2.16) and S (0.34 to 0.91) in the stationary phase, along with marked variations of C and N fractions among isolates in the exponential phase. The cell C (5.7 to 203.7 pg) and N (0.65 to 6.1 pg) content exhibited a significant ( p < 0.001) linear relationship with the cell volume (27.7 to 510 μm 3 ). On further analysis of the relationship across the two growth phases, we found the equation (cell C (pg) = 0.356 × cell volume (μm 3 ) + 20.922) for stationary phase cells more appropriate for C estimation of natural thraustochytrids. This study provides the first experimental evidence of higher cell C density than the current estimate and relatively larger C contribution of thraustochytrids than bacteria to the marine organic pool.

Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The interest in bioactive compounds from microalgae is increasing since they have medicinal and nutritional areas. The present work aims to evaluate the potential pharmaceutical interest of extracts from three eustigmatophyte strains from the Coimbra Collection of Algae (ACOI): Chlorobotrys gloeothece, Chlorobotrys regularis and Characiopsis aquilonaris. Antioxidant and antiproliferative activities were determined as well as chlorophyll a, carotenoid and phenolic total contents. In addition, major pigments and sterols were identified and quantified. The three strains were grown until the stationary phase and then the biomass was extracted. Antioxidant activity was measured by TEAC, DPPH and FRAP assays and antiproliferative effect was assessed by the MTT method on MCF-7, PC-3 and NHDF cells. The pigment and phenolic total contents were determined by spectrophotometry. Of these strains, C. aquilonaris showed the highest antioxidant activity measured by TEAC and FRAP assays (23.98 ± 0.01 μmol TE eq g -1 DW and 42.57 ± 0.04 μmol TE eq g -1 DW, respectively), a selective effect in reduting MCF-7 cells proliferation and a larger amount of chlorophyll a, carotenoids and phenolic content (18.40 ± 0.00 μg chlorophyll a mg -1 DW, 2.27 ± 0.00 mg carotenoids g -1 DW and 6.23 ± 0.01 mg GAE g -1 DW, respectively). A positive correlation between chlorophyll a and TEAC assay was observed, as well as between carotenoids and TEAC and FRAP assays, suggesting these compounds as important contributors to significant antioxidant activity. Violaxanthin, cholesterol and stigmasterol were present in larger amount in C. aquilonaris while C. regularis showed a higher amount of β-carotene. These results suggest that these three ACOI eustigmatophytes are promising for applications in the improvement of human health, particularly in cancer prevention and treatment.

Microalgal oil is considered a promising candidate for edible oils. However, investigation of the refining processes of microalgal oil has been limited, especially deacidification. In this work, microcrystalline cellulose (MCC) was pretreated using different methods and utilized for the first time in the deacidification of microalgal oil. Detection results from FTIR and XRD indicated alkali pretreatment had a significant effect on the structure of MCC. Some inter- and intramolecular hydrogen bonds in AMCC (alkali-pretreated MCC) were destroyed, and crystallinity index of cellulose decreased, which increased its adsorption capacity and the reaction of OH groups with free fatty acids. Some NaOH was adsorbed into AMCC through cellulose swelling, which also contributed to deacidification. The interaction with oil was also improved with many cracks and voids on the surface of AMCC. AMCC could reduce the acid value to about 2 mg KOH/g. Comparatively, original MCC and MCC pretreated with microwave or ultrasound did not exhibit the ability to deacidify. Furthermore, the conditions of alkali treatment were optimized. Treatment with 20% NaOH for 20 min was optimal. Compared with other adsorbents, such as sodium silicate and chitosan treated with alkali and resin, only AMCC could effectively reduce acid value while maintaining high lipid recovery. Therefore, AMCC was considered a better adsorbent for the deacidification of microalgal oil.

Photosynthetic organisms have diversified light-harvesting complexes (LHCs) to collect solar energy efficiently, leading to an acquisition of their ecological niches. Herein we report on biochemical and spectroscopic characterizations of fucoxanthin chlorophyll a/c-binding protein (FCP) complexes isolated from a marine pinguiophyte Glossomastix chrysoplasta. The pinguiophyte FCP showed one subunit band in SDS-PAGE and one protein-complex band with a molecular weight at around 66 kDa in clear-native PAGE. By HPLC analysis, the FCP possesses chlorophylls a and c, fucoxanthin, and violaxanthin. To clarify excitation-energy-relaxation processes in the FCP, we measured time-resolved fluorescence spectra at 77 K of the FCP adapted to pH 5.0, 6.5, and 8.0. Fluorescence curves measured at pH 5.0 and 8.0 showed shorter lifetime components compared with those at pH 6.5. The rapid decay components at pH 5.0 and 8.0 are unveiled by fluorescence decay-associated (FDA) spectra; fluorescence decays occur in the 270 and 160-ps FDA spectra only at pH 5.0 and 8.0, respectively. In addition, energy-transfer pathways with time constants of tens of picoseconds are altered under the basic pH condition but not the acidic pH condition. These findings provide novel insights into pH-dependent energy-transfer and energy-quenching machinery in not only FCP family but also photosynthetic LHCs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Fucoxanthin chlorophyll-binding proteins (FCPs) are the major light-harvesting complexes of diatoms. In this work, FCPs isolated from Cyclotella meneghiniana have been studied by means of optically detected magnetic resonance (ODMR) and time-resolved electron paramagnetic resonance (TR-EPR), with the aim to characterize the photoprotective mechanism based on triplet-triplet energy transfer (TTET). The spectroscopic properties of the chromophores carrying the triplet state have been interpreted on the basis of a delved analysis of the recently solved crystallographic structures of FCP. The results point toward a photoprotective role for two fucoxanthin molecules exposed to the exterior of the FCP monomers. This shows that FCP has adopted a structural strategy different from that of related light-harvesting complexes from plants and other microalgae, in which the photoprotective role is carried out by two highly conserved carotenoids in the interior of the complex., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein-protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named "Applied Proteomics" has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer-the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named "applied proteomics". It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana , the bioalgorithm identified over 400 proteins with potential uses; one of them was selected as UCA01, "in vitro" and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined.

Along with the daily growth of the market requirements for docosahexaenoic acid (DHA) algae oil, a large DHA ingredients are needed to ensure worldwide supply. Undoubtedly a high-productive strain would be the prerequisite for high quality and yield. A comprehensive understanding of the processes of DHA synthesis from glycolysis to the lipid accumulation would be benefit to achieve the final optimization of DHA production. In this study, we comprehensively characterized the metabolic profiles of a Schizochytrium sp. strain, which has higher DHA content and different biomass amino acid composition compared with the wild type to explore the affected pathways and underlying mechanism. Combined with the multivariate statistical analysis, twenty-two differential metabolites were screened as relevant to the discrepancy between two strains. The results showed relatively downregulated glycolysis and saturated fatty acids (SFA) synthesis, and upregulated TCA cycle, amino acids and polyunsaturated fatty acids (PUFA) synthesis in DHA high yield strain. The current study provide a terminal picture of gene regulation from downstream metabolism and demonstrate the advantage of metabolomics in characterizing metabolic status which in turn could provide effective information for the metabolic engineering., (Copyright © 2020. Published by Elsevier B.V.)

The current work was planned to assess the effects of dietary microalga, Nannochloropsis oculata (NP) on the growth indices, serum biochemistry, non-specific immunity, and resistance of Nile tilapia (Oreochromis niloticus) juveniles against challenge with pathogenic Aeromonas veronii. Fish (10.21 ± 0.28 g) were randomly divided into four treatments in triplicates and were fed on diets supplemented with different levels of NP (0, 5, 10, and 15%) for eight weeks. The results showed that the dietary 5% NP significantly improved the growth parameters (final body weight, weight gain, specific growth rate, and total feed intake) (P < 0.05). Meanwhile, the feed conversion ratio was significantly decreased in NP-supplemented groups with respect to the control group. The serum protein profile (total protein, albumin, globulin, and albumin/globulin ratio) was significantly decreased by 15% NP supplemented group. Meanwhile, liver enzymes (alanine transaminase, and aspartate transaminase) activities were not significantly affected by NP dietary supplementation (P < 0.05). Serum lysozyme activity, nitric oxide (NO), and nitroblue tetrazolium (NBT) levels were significantly enhanced only in 5% NP supplemented group (P < 0.05). Interestingly, the hepatopancreatic and intestinal tissues had apparently normal histomorphology of Nile tilapia fed at 5% NP-supplemented diets. Significant upregulation of cytokines [interleukin 1beta (IL-1β), interleukin 8 (IL-8), interferon-gamma (IFN-γ), transforming growth factor-beta (TGF- β), and tumor necrosis factor-alpha (TNF-α)], with significant downregulation of the antioxidant gene [superoxide dismutase (SOD)] in 10% and 15% NP supplemented groups. Attractively, the relative level of protection (RLP) against challenge with pathogenic A. veronii was significantly elevated in 10% and 15% NP supplemented groups. Conclusively, the obtained results reflect the beneficial roles of dietary NP to improve growth and boost the immune responses of Nile tilapia., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mixotrophic cultivation can increase microalgae productivity, yet the associated lipid metabolism remains mostly unknown. Stable isotope labeling was used to track assimilation of glycerol into the triacylglyceride (TAG) and membrane lipids of Nannochloropsis salina. In N-replete media, glycerol uptake and 13 C incorporation into acyl chains were, respectively, 6-fold and 12-fold higher than in N-deplete conditions. In N-replete cultures, 42% of the carbon in the consumed glycerol was assimilated into lipid acyl chains, mostly in membrane lipids rather than TAG. In N-deplete cultures, only 11% of the limited amount of consumed glycerol was fixed into lipid acyl chains. Labeled lipid-associated glycerol backbones were predominantly 13 C 3 labeled, suggesting that intact glycerol molecules were directly esterified with fatty acids/polar head groups. However, the presence of singly and doubly labeled lipid-bound glycerol species suggested that some glycerol also went through the central carbon metabolism before forming glycerol-3-phosphate destined for lipid esterification. 13 C incorporation was higher in the saturated and monounsaturated than the polyunsaturated acyl chains of TAG, indicating the flux of carbon from glycerol went first to de novo fatty acid synthesis before acyl editing reactions. The results demonstrate that nitrogen availability influences both glycerol consumption and utilization for lipid synthesis in Nannochloropsis, providing novel insights for developing mixotrophic cultivation strategies., (© 2020 Wiley Periodicals LLC.)

Immunostimulants are key molecules in aquaculture since they heighten defensive responses and protection against pathogens. The present study investigated the treatment of Senegalese sole larvae with a whole-cell crude extract of the microalgae Nannochloropsis gaditana (Nanno) and programming of growth and the immune system. Larvae at hatch were treated with the Nanno extracts for 2 h and thereafter were cultivated for 32 days post-hatch (dph) in parallel with an untreated control group (CN). Dry weight and length at 21 days post-hatch (dph) were higher in post-larvae of the Nanno than CN group. These differences in weight were later confirmed at 32 dph. To evaluate changes in the immune response associated with Nanno-programming treatments, the Nanno and CN post-larvae were supplied with two bioactive compounds yeast β-glucan (Y) and a microalga extract from the diatom Phaeodactylum tricornutum (MAe). The bioactive treatments were administrated to the treatment groups through the live prey (artemia metanauplii, 200 artemia mL -1 ) enriched for 30 min with MAe or Y (at 2 mg mL -1  SW) or untreated prey in the case of the negative control (SW). The effect of the treatments was assessed by monitoring gene expression, enzyme activity and mortality over 48 h. The post-larvae sole supplied with the bioactive compounds Y and MAe had increased mortality at 48 h compared to the SW group. Moreover, mortality was higher in Nanno-programmed than CN post-larvae. Lysozyme and total anti-protease enzymatic activities at 6 and 24 h after the start of the trial were significantly higher in the Nanno and MAe supplied post-larvae compared to their corresponding control (CN and SW, respectively). Immune gene transcripts revealed that il1b, cxc10 and mx mRNAs were significantly different between Nanno and CN post-larvae at 6 and 24 h. Moreover, the expression of il1b, tnfa, cxc10, irf3, irf7 and mx was modified by bioactive treatments but with temporal differences. At 48 h after bioactive treatments, Y and SW post-larvae were challenged with the lymphocystis disease virus (LCDV). No difference existed in viral copy number between programming or bioactive treatment groups at 3, 6 and 24 h after LCDV challenge although the total number of copies reduced with time. Gene expression profiles in the LCDV-challenged group indicated that post-larvae triggered a wide defensive response compared to SWC 24 h after challenge, which was modulated by programming and bioactive compound treatments. Cluster analysis of expressed genes separated the SW and Y groups indicating long-lasting effects of yeast β-glucan treatment in larvae. A noteworthy interaction between Nanno-programming and Y-treatment on the regulation of antiviral genes was observed. Overall, the data demonstrate the capacity of microalgal crude extracts to modify sole larval plasticity with long-term effects on larval growth and the immune responses., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nannochloropsis oceanica represents a preferred oleaginous alga for producing lipids. Here we found that phosphorus deprivation (PD) caused a severe decrease in protein and a considerable increase in lipids including triacylglycerol (TAG), yet it had little effect on the carbohydrate level and biomass production of N. oceanica . The combinatorial analysis by integrating physiological, biochemical, and transcriptomic data unraveled the molecular mechanisms underlying PD-induced lipid accumulation. Albeit attenuating the Calvin-Benson cycle, PD stimulated the C4-like pathway to maintain CO 2 fixation for biomass production. PD attenuated nitrogen utilization and enhanced protein catabolism thus leading to protein decrease, from which the carbon was likely salvaged into the stimulated tricarboxylic acid cycle for supplying lipid synthesis with carbon precursors. The impairment of TAG catabolism by downregulating certain lipases rather than the stimulation of TAG assembly pathways contributed to PD-boosted TAG increase. These findings provide novel insights into PD-induced lipogenesis without compromising biomass production by N. oceanica .

Biochemical food quality constraints affect the performance of consumers and mediate trait variation among and within consumer species. To assess inter- and intraspecific differences in fatty acid retention and conversion in freshwater rotifers, we provided four strains of two closely related rotifer species, Brachionus calyciflorus sensu stricto and Brachionus fernandoi , with food algae differing in their fatty acid composition. The rotifers grazed for 5 days on either Nannochloropsis limnetica or Monoraphidium minutum , two food algae with distinct polyunsaturated fatty acid (PUFA) profiles, before the diets were switched to PUFA-free Synechococcus elongatus , which was provided for three more days. We found between- and within-species differences in rotifer fatty acid compositions on the respective food sources and, in particular, highly specific acclimation reactions to the PUFA-free diet. The different reactions indicate inter- but also intraspecific differences in physiological traits, such as PUFA retention, allocation and bioconversion capacities, within the genus Brachionus that are most likely accompanied by differences in their nutritional demands. Our data suggest that biochemical food quality constraints act differently on traits of closely related species and of strains of a particular species and thus might be involved in shaping ecological interactions and evolutionary processes. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

For the last two decades there has been a rise in awareness about the general low dietary intake of vitamin D 3 . Fish have the highest natural content of vitamin D 3 , which is suggested to originate from zooplankton and microalgae. However there are no studies reporting which microalgal species may be the source of vitamin D 3 . In this study, four selected microalgal species were cultivated during exposure of artificial UVB. The effect of UVB dose on the growth and biochemical composition of the cells (vitamin D 3 , PUFAs and carotenoids) was evaluated. Of the four species, exclusively Nannochloropsis oceanica was able to produce vitamin D 3 (up to 1 ± 0.3 µg/g DM), and production was significantly enhanced by increasing the dose of the UVB. These findings suggest that N.oceanica exposed to artificial UVB could be used as a new natural source of vitamin D 3, either as direct source or through animal feed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The global bivalve shellfish industry makes up 25% of aquaculture, is worth USD $17.2 billion year -1 , and relies upon a supply of juvenile bivalves produced by adult broodstock in hatcheries. Today large quantities of live algae are grown to feed broodstock at $220 kg -1 , driving highly unsustainable energy and resource use. New advances in algal and microencapsulation technology provide solutions. We developed microencapsulated Schizochytrium algae diets, which can be produced sustainably at < $2 kg -1 from organic side-streams, and are shelf-stable to minimise waste. Physiological, histological, and cutting-edge metabolomic analyses demonstrate that in commercial settings sustainable microencapsulated diets facilitate improved sexual development and 12 × greater omega-3 levels in oysters relative to conventional live algal diets. Every tonne bivalve protein produced instead of fish spares 9 ha, 67 tonnes CO 2 , and 40,000 L freshwater. Further research into microencapsulated diets could support bivalve industry expansion, and contribute towards a step-change in sustainable global food production through improved aquaculture practices.

Light oxygen voltage-sensing (LOV) domains are widely found in photoreceptor proteins of plants, algae, fungi, and bacteria. Structural studies of LOV domains suggest that Phe and Gln residues located in the proximity of the chromophore undergo conformational changes upon illumination; however, the molecular mechanism associated with activation of the effector domain remains to be elucidated. Photozipper (PZ) protein is an N-terminally truncated aureochrome-1 comprising a LOV domain and a basic leucine zipper domain. Blue light (BL) induces PZ dimerization and subsequently increases its affinity for target DNA. In this study, we prepared PZ mutants with substitutions of F298 and Q317 and performed quantitative analyses in dark and light states. Substitutions of Q317 significantly reduced the light-induced changes in PZ affinity for the target DNA, especially in the case of the high affinities observed in the dark state. Upon illumination, all PZ mutants showed increased affinity for the target sequence, which demonstrated a clear correlation with the dimer fraction of each PZ mutant. These results suggest the existence of a conformational equilibrium and that its shift by a synergistic interaction between the chromophore and protein moiety probably enables BL-regulated switching of aureochrome-1.

The purpose of this paper was to evaluate the toxicological potential of a heterotrophically grown unextracted Aurantiochytrium limacinum biomass (AURA) when used as a food additive. The following toxicological assessments were conducted on this novel docosahexaenoic acid rich feed ingredient: Mutagenic activity was tested by means of the Ames' test using five Salmonella typhimurium strains; clastogenic activity was investigated using the micronucleus test in male and female Sprague Dawley rats; genotoxic activity was assessed by means of the in vitro metaphase analysis tests in human lymphocytes; oral toxicity was tested by administration of AURA at various concentrations; eye and skin irritation was assessed in rabbits according to OECD guidelines; skin sensitivity was established in guinea pigs by means of the Buehler test. All conducted tests were considered valid under the experimental conditions. No significant mutagenic activity or clastogenic activity was observed. Genotoxic activity in human lymphocytes was not induced. Oral administration of 276 mg AURA/kg bw 1 and 2000 mg AURA/kg bw resulted in no mortality or signs of acute toxicity. Daily administration of 1000 mg AURA/kg bw caused no mortality or biologically relevant signs of toxicity and was established as the No Observable Adverse Effect Level. AURA was also found to be a non-irritant for the eye and skin of the rabbit and was non-sensitizing to guinea pig skin., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors, G.P. Dillon, J.D. Keegan and C. A. Moran are employees of Alltech which produced and marketed ALL-G-RICH®, the commercial product used in this study., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The aim of this trial is to assess the effect of Almega ® PL on improving the Omega-3 Index, cardio-metabolic parameters, and other biomarkers in generally healthy individuals. The benefits of long-chain omega-3 fatty acids for cardiovascular health are primarily built upon mixtures of docosahexaenoic (DHA) and eicosapentaenoic acids (EPA). Highly purified EPA therapy has proven to be particularly effective in the treatment of cardiovascular disease, but less is known about the benefits of EPA-only supplementation for the general healthy population. Almega ® PL is a polar rich oil (>15%) derived from the microalga Nannochloropsis that contains EPA (>25%) with no DHA. Participants ( n = 120) were given a capsule of 1 g/day of either Almega ® PL or placebo for 12 weeks. Differences in the Omega-3 Index, cardiometabolic markers, and other general health indicators were measured at the baseline, six, and 12 weeks. Compared to the placebo group, Almega ® PL supplementation significantly increased the Omega-3 Index and EPA concentration from 4.96 ± 0.90 and 0.82 ± 0.37% at the baseline to 5.75 ± 0.90 and 1.27 ± 0.36 at week 12, respectively. Very-low-density lipoprotein cholesterol (VLDL) decreased by 25%, which resulted in a significant decrease in total cholesterol compared to the placebo. Interestingly, the decrease in VLDL was not associated with an increase in LDL, which seems to be a benefit associated with EPA-only based formulations. Collectively, these results show that Almega ® PL provides a natural EPA-only option to increase EPA and manage cholesterol levels in the general population.

Non-alcoholic fatty liver disease represents the most common liver disease and is characterized by an excess of lipid accumulation in hepatocytes, mainly stored as triglycerides. Phaeodactylum tricornutum is a marine microalga, which is rich in bioactive molecules known to be hepatoprotective, such as n -3 long-chain polyunsaturated fatty acids and fucoxanthin. The aim of this study was to investigate the effects of a carotenoid extract from P. tricornutum in a cellular model of non-alcoholic fatty liver disease induced by palmitate treatment. The combined effects of carotenoids and lipids, especially n -3 long-chain polyunsaturated fatty acids, were also investigated by using a total lipophilic extract. HepG2 cells were exposed for 24 h to 250 µM palmitate with or without the addition of carotenoid extract (6 μg/mL) or total lipophilic extract (100 μg/mL). The addition of carotenoid extract or total lipophilic extract prevented the accumulation of triglycerides, total cholesterol and cholesterol esters. The carotenoid extract and total lipophilic extract also decreased the mRNA expression levels of genes involved in lipogenesis ( ACACA , FASN , SCD and DGAT1 ) and cholesterol esterification ( ACAT1 / SOAT1 ). In addition, the total lipophilic extract also downregulated the LXR/NR1H3 and SREBF1 genes, which are involved in lipogenesis regulation. By contrast, the carotenoid extract increased the mRNA level of CPT1A , a β-oxidation related gene, and reduced the lipid droplet accumulation. In conclusion, this study highlights the preventive effects against non-alcoholic fatty liver disease of the two microalga extracts.

Aims: The effect of algae and its extract supplementation on glycolipid metabolism has not been finalized. Therefore, the purpose of the meta-analyses was to assess the effects of its supplementation on glycolipid metabolism concentration., Methods: We have systematically searched PubMed, Web of Science, the Cochrane Library and Embase to identify randomized controlled trials (RCTs) that investigated the impact of algae and its extracts supplementation on glycolipid metabolism. Effect size analysis was performed using weighted mean difference (WMD) and 95% CI between the methods of the experiment group and the control group. Subgroup analyses were performed to explore the possible influences of study characteristics. Publication bias and sensitivity analysis were also performed., Results: A total of 27 RCTs (31 trials) with 1221 participants were finally selected for the meta-analysis. The algae and its extract intervention significantly decreased glycosylated hemoglobin (HbA1c, WMD = -0.18%; 95% CI: -0.27 to -0.10; p < 0.001), high-density lipoprotein cholesterol (HDL-C, WMD = -0.22 mmol/L; 95% CI: -0.38 to -0.06; p = 0.008), and triglycerides (TC, WMD = -0.31 mmol/L; 95% CI: -0.37 to -0.25; p < 0.001) levels and increased insulin (WMD = 6.05 pmol/mL; 95% CI: 4.01 to 8.09; p < 0.001) levels. It did not significantly change the blood glucose, homeostasis model assessment-insulin resistance index (HOMA-IR), 2-h post-meal blood glucose (2hPBG) and other lipid profiles. Subgroup analyses based on the duration of intervention and subjects demonstrated that the intervention of algae and its extracts for 10 weeks or fewer and more than 40 subjects decreased TC levels ( p < 0.05). Moreover, the intervention reduced TC and 2hPBG concentrations for East Asians ( p < 0.05)., Conclusions: Our findings provided evidence that algae and its extract interventions were beneficial for the regulation of human glycolipid metabolism. More precise RCTs on subjects are recommended to further clarify the effect of algae, seaweed polysaccharide, seaweed polypeptide, algae polyphenol and its products intervention on glycolipid metabolism.

Effect of water accommodated fractions (WAF) of #180 fuel oil on fixed carbon and nitrogen in microalgae was studied by stable isotopes. Platymonas helgolandica, Heterosigma akashiwo and Nitzschia closterium were exposed to five WAF concentrations for 96 h. The δ 13 C value of microalgae was significantly lower than that of the control group, indicated that carbon was limited in the WAF concentrations. The δ 13 C value of microalgae appeared peak valley at 48 h in control group, corresponding to the enhanced capacity in carbon fixation during microalgae photosynthesis. The physiological acclimation capacity of microalgae was revealed by the occurrence time when the δ13C value was in peak valley, and thus the physiological acclimation capacity of microalgae decreased in the order of Nitzschia closterium > Heterosigma akashiwo > Platymonas helgolandica. Principal component analysis (PCA) were applied to the δ13C value in order to verify the "hormesis" phenomenon in microalgae. The δ13C value could discriminate between stimulatory effects at low doses and inhibitory effects at high doses. In addition, the present study also investigated the effect of the nitrogen on microalgae growth. Because microalgae could still absorb the NO 3 -N and release of NO 2 -N and NH 4 -N in present study, the nitrogen cycle in microalgae was in the equilibrium status. The δ 15 N value in microalgae exhibited no obvious change with the increasing of WAF concentrations at the same time. However, due to the enrichment of nitrogen, the δ 15 N value first increased gradually with the time and finally was stable. Overall, the fractionation of carbon and nitrogen stable isotopes illustrated that the effect of carbon on the growth of microalgae was more prominent than nitrogen. Stable isotopes was used to investigate the influence of WAF on fixed carbon and nitrogen in microalgae growth, providing a fundamental theoretical guidance for risk assessment of marine ecological environment., Competing Interests: Declaration of competing interest Submission of an article implies that the work described has not been published previously (except in the form of an abstract or as part of a published lecture or academic thesis), that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, without the written consent of the copyright-holder. By attaching this declaration to the submission, the corresponding author certifies that:, (Copyright © 2020 Elsevier Inc. All rights reserved.)

The objective of this work was to evaluate the effects of extrusion pretreatment on the efficiency of lipid extraction and the quality of lipid extracted from the microalga Nannochloropsis oceanica. Five processing parameters of extrusion pretreatment using a twin-screw extruder were studied: die configuration, distance from the internal surface of the die to the end of the screw (δ), temperature, screw rotation speed, and the water content of biomass. The results showed that die configuration, δ, and the water content of the biomass were vital factors affecting lipid extraction efficiency. After the pretreatment of extrusion, the extraction time was shortened from 10 h to 20 min, and the amount of solvent used was reduced to one-third of that without pretreatment. The content of polyunsaturated fatty acid (PUFA) in crude lipid increased by over 30 percent of those components without pretreatment, especially for eicosapentaenoic acid (EPA), which increased by 37.45%. In addition, the unsaponifiable content and acid value of the crude lipid extracted from the extruded microalga decreased significantly, which would facilitate further refining processes.

Coagulation and flocculation can remove particulate algal cells effectively; however, they are not very effective for removing dissolved algal organic matter (AOM) in drinking water plants. In this work, optimum coagulation conditions using alum for both extracellular and intracellular organic matter of six different algal and cyanobacterial species were determined. Different coagulation conditions such as alum dosage, pH, and initial dissolved organic carbon (DOC) were tested. Hydrophobicity, hydrophilicty, and transphilicity of the cellular materials were determined using resin fractionation method. The removal of DOC by coagulation correlated well with the hydrophobicity of the AOM. The disinfection by-product formation potential (DBPFP) of various fractions of AOM was determined after coagulation. Although, higher removal occurred for hydrophobic AOM during coagulation, specific DBPFP, which varied from 10 to 147 μg/mg-C was higher for hydrophobic AOM. Of all the six species, highest DBPFP occurred for Phaeodactylum tricornutum, an abundant marine diatom species, but is increasingly found in surface water., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Impact of feeding n-3 fatty acids (FA) to ISA brown and Shaver white breeders and their progeny on bone development in pullets was investigated. Breeders were fed Control (CON); CON + 1% microalgae (DMA: Aurantiochytrium limacinum) as the source of docosahexaenoic acid; and CON + 2.6% of a co-extruded mixture of full-fat flaxseed (FFF) and pulses mixture as source of α-linolenic acid. Test diets (DMA and FFF) were balanced for total n-3 FA and n-6: n-3 FA ratio. Samples of day-old progeny were euthanized for bone mineral content (BMC) and tibia collagen type II. The remaining pullets were fed posthatch treatments as follows: from breeder CON: CON (CON-CON), DMA (CON-DMA), and FFF (CON-FFF), from breeder DMA: CON (DMA-CON) and DMA (DMA-DMA) and from breeder FFF: CON (FFF-CON) and FFF (FFF-FFF). A total of 60 pullets per posthatch diets were reared in cages (12 pullets/cage, n = 5) with free access to feed and water, bled at 6, 12, and 18 wk of age (WOA) for bone turnover markers and necropsied at 18 WOA for tibia and femur samples. Day-old pullets from breeder fed CON had greater BMC (P < 0.001) relative to those from breeders fed other diets. There was strain and diet interaction (P ≤ 0.024) on tibia breaking strength (TBS) and tibia cortical ash concentration at 18 WOA such that diet responses were only observed in Shaver white pullets. In this context, TBS of DMA-DMA and FFF-FFF was greater than for pullets originating from CON breeder, and the cortical ash weight of DMA-DMA and FFF-FFF pullets was 23.8 and 20.2%, respectively, higher than for CON-CON pullets. In conclusions, the strain effects were strong on tibia attributes on 18-week-old pullets. Breeder feeding of n-3 FA was more effective when concomitant with posthatch feeding of n-3 FA in supporting the skeletal strength and cortical bone development in Shaver white pullets. Further investigations are warranted to establish the impact these strategies on skeletal health during laying cycle., (Copyright © 2019. Published by Elsevier Inc.)

Mangrove sediments represent unique microbial ecosystems that act as a buffer zone, biogeochemically recycling marine waste into nutrient-rich depositions for marine and terrestrial species. Marine unicellular protists, thraustochytrids, colonizing mangrove sediments have received attention due to their ability to produce large amounts of long-chain ω3-polyunsaturated fatty acids. This paper represents a comprehensive study of two new thraustochytrids for their production of valuable biomolecules in biomass, de-oiled cakes, supernatants, extracellular polysaccharide matrixes, and recovered oil bodies. Extracted lipids (up to 40% of DW) rich in polyunsaturated fatty acids (up to 80% of total fatty acids) were mainly represented by docosahexaenoic acid (75% of polyunsaturated fatty acids). Cells also showed accumulation of squalene (up to 13 mg/g DW) and carotenoids (up to 72 µg/g DW represented by astaxanthin, canthaxanthin, echinenone, and β-carotene). Both strains showed a high concentration of protein in biomass (29% DW) and supernatants (2.7 g/L) as part of extracellular polysaccharide matrixes. Alkalinization of collected biomass represents a new and easy way to recover lipid-rich oil bodies in the form of an aqueous emulsion. The ability to produce added-value molecules makes thraustochytrids an important alternative to microalgae and plants dominating in the food, pharmacological, nutraceutical, and cosmetics industries.

There is limited information on feeding egg-type chick breeders n-3 polyunsaturated fatty acids (PUFA) and its impact on hatching egg quality and embryonic fatty acid (FA) utilization. We investigated the effects of feeding brown and white egg-type chick breeders diets containing sources of n-3 PUFA on egg composition, apparent embryonic FA utilization, and intestinal FA transporter in hatchlings. Twenty-six-week-old ISA brown and Shaver white breeders were fed either 1) control (CON); 2) CON + 1% of microalgae (DMA, Aurantiochytrium limacinum) fermentation product, as a source of docosahexaenoic acid (DHA); or 3) CON + 2.60% of coextruded full-fat flaxseed and pulse mixture (FFF, 1:1 wt/wt) as a source of α-linolenic acid (ALA). Test diets had similar total n-3 and n-6:n-3 ratio. Eggs were hatched, and residual yolk (RY) samples taken for FA analyses. Apparent embryonic FA utilization was calculated by subtracting concentration of FA in RY from concentration of FA in yolk before incubation. There was an interaction between strains and diets (P < 0.05) on DHA in phospholipid and triglyceride fractions of yolk. Both n-3 PUFA sources increased DHA to a greater extent in Shaver white than in ISA brown. The interactive effect of strains and diets (P = 0.019) on embryonic utilization of ALA was such that DMA and FFF reduced ALA utilization, and this pattern was more prevalent in Shaver white birds than in ISA brown birds. There was no interaction between strains and diets on DHA utilization (P > 0.05). Embryos from hens fed n-3 PUFA sources used less total FA in phospholipid fraction (P < 0.001), and they preferentially used more DHA than CON embryos. Shaver white embryos used more (P < 0.05) ALA and DHA than ISA brown embryos. Although data suggested Shaver white had higher propensity of depositing DHA than ISA brown, irrespective of strain, feeding n-3 PUFA modified embryonic pattern of FA utilization toward utilization of DHA., (Copyright © 2019. Published by Elsevier Inc.)

Background: The research deals with the addition of microalgae Schizochytrium limacinum as an alternative to fish oil in a feed-mixture for laying-hens and its effect on the deposition of n-3 polyunsaturated fatty acids (n-3-PUFAs) [α-linoleic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in the lipids of egg yolks. In the study, 240 Tetra SL laying hens housed in enriched cages were used, divided into six groups, each in five repetitions. Groups E1, E3 and E5 were given 0.5%, 1.0% and 1.5% microalgae and groups E2, E4 and E6 were given feed mixtures with 0.5%, 1.0% and 1.5% fish oil. The mixtures were modified on the basis of 17% of the crude protein and 11.7 MJ ME kg -1 ., Results: The results showed satisfactory disposal of n-3 PUFA in egg yolks of laying-hens fed mixtures with the addition of either fish oil or microalgae. Eggs of E1, E3 and E5 groups contained in 100 g: 321.07 mg, 361.60 mg and 399.34 mg n-3 PUFA, respectively (P < 0.001). Eggs of E2, E4 and E6 groups contained in 100 g: 346.25 mg, 346.17 mg and 369.02 mg n-3 PUFA, respectively (P < 0.001). By increasing the content of fish oil or microalgae in feed-mixtures for laying hens, the ratio of n-6/n-3 PUFA in egg yolk lipids (P < 0.001) was decreased., Conclusion: Our results justified the usage of the microalgae Schizochytrium limacinum in the enrichment of table eggs with n-3 fatty acids as an alternative feed to fish oil. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

The transport of hydrophobic compounds to recipient cells is a critical step in nutrient supplementation. Here, we tested the effect of phospholipid-based emulsification on the uptake of hydrophobic compounds into various tissue culture cell lines. In particular, the uptake of ω-3 fatty acids from micellar or nonmicellar algae oil into cell models for enterocytes, epithelial cells, and adipocytes was tested. Micellization of algae oil did not result in adverse effects on cell viability in the target cells. In general, both micellar and nonmicellar oil increased intracellular docosahexaenoic acid (DHA) levels. However, micellar oil was more effective in terms of augmenting the intracellular levels of total polyunsaturated fatty acids (PUFAs) than nonmicellar oil. These effects were rather conserved throughout the cells tested, indicating that fatty acids from micellar oils are enriched by mechanisms independent of lipases or lipid transporters. Importantly, the positive effect of emulsification was not restricted to the uptake of fatty acids. Instead, the uptake of phytosterols from phytogenic oils into target cells also increased after micellization. Taken together, phospholipid-based emulsification is a straightforward, effective, and safe approach to delivering hydrophobic nutrients, such as fatty acids or phytosterols, to a variety of cell types in vitro. It is proposed that this method of emulsification is suitable for the effective supplementation of numerous hydrophobic nutrients., Competing Interests: The authors declare no conflicts of interest. PM International AG provided support in the form of the salaries for M.I. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The ability to identify drivers responsible for algal community shifts is an important aspect of environmental issues. The lack of long-term datasets, covering periods prior to these shifts, is often limiting our understanding of drivers responsible. The freshwater alga, Gonyostomum semen (Raphidophyceae), has significantly increased distribution and mass occurrences in Scandinavian lakes during the past few decades, often releasing a skin irritating slime that causes discomfort for swimmers. While the alga has been extensively studied, long-term data from individual lakes are often absent or greatly limited and drivers behind this species' success are still not clear. However, if specific and persistent taxa biomarkers for G. semen could be detected in dated sediment cores, long-term data would be improved and more useful. To test for biomarkers, we examined the pigment composition of several G. semen strains in culture. Further, dated sediment core samples from Lake Lundebyvann, Norway, were used to test the pigments' suitability as biomarkers in paleolimnological studies. Modifications to a common analysis allowed for the successful detection of the polar xanthophyll heteroxanthin and the non-polar chlorophyll a, as well as several other algal pigments by using high performance liquid chromatography-photometric diode arrays (HPLC-PDA). Heteroxanthin was confirmed by liquid chromatography-mass spectrometry (LC-MS) and detected by HPLC-PDA in all examined G. semen strains, along with chlorophyll a. Using HPLC-PDA, we also identified and confirmed the presence of the biomarker, xanthophyll heteroxanthin, in sediment core samples up to 60 years of age. The specificity of this xanthophyll was also tested by examining a wide range of algal strains from common Norwegian phytoplankton species. Heteroxanthin was not detected in any species commonly occurring in significant amounts in Norwegian lakes. We therefore conclude that heteroxanthin is a suitable pigment biomarker for G. semen and that this pigment can be successfully used for paleolimnological studies., Competing Interests: The authors have declared that no competing interests exist.

Lipids produced from agricultural and industrial residues using oleaginous microorganisms for use as biofuels are attracting the attention of researchers due to their environmental benefits. However, low efficiencies and high costs limit their application to a certain extent. The present study is the first to use inositol as an enhancer to improve the production and accumulation of lipids during fermentation by the microalga Schizochytrium sp. SR21. The study aimed to maximize the production of lipids and docosahexaenoic acid (DHA) by optimizing the conditions of inositol addition into the fermentation medium. The corresponding key enzyme and metabolite profiles of SR21 were evaluated. The results indicated that the addition of 120 mg L -1 of inositol to the medium at 48 h improved lipid and DHA production by 13.90 and 20.82%, resulting in total concentrations of 22.86 and 8.53 g/L, respectively. Moreover, the ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs) increased by 23.38% and is consistent with the results of the metabolomic analysis. The activity of enzymes (i.e., PC, G6PDH, NADPH-ME, and ACL) related to fatty acid synthesis in strain SR21 also increased significantly (43.38%, 28.68%, 37.47%, and 19.87%, respectively). Metabolomic analysis also showed that inositol promoted lipid synthesis in SR21 and significantly increased the relative proportion of UFAs by affecting the citrate cycle and SFA and UFA metabolic pathways. Thus, inositol is an ideal enhancer of lipid production and accumulation by oleaginous microorganisms. Graphical abstract.

Background: Schizochytrium has been widely used in industry for synthesizing polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA). However, unclear biosynthesis pathway of PUFAs inhibits further production of the Schizochytrium. Unsaponifiable matter (UM) from mevalonate pathway is crucial to cell growth and intracellular metabolism in all higher eukaryotes and microalgae. Therefore, regulation of UM biosynthesis in Schizochytrium may have important effects on fatty acids synthesis. Moreover, it is well known that UMs, such as squalene and β-carotene, are of great commercial value. Thus, regulating UM biosynthesis may also allow for an increased valuation of Schizochytrium., Results: To investigate the correlation of UM biosynthesis with fatty acids accumulation in Schizochytrium, fluconazole was used to block the sterols pathway. The addition of 60 mg/L fluconazole at 48 h increased the total lipids (TLs) at 96 h by 16% without affecting cell growth, which was accompanied by remarkable changes in UMs and NADPH. Cholesterol content was reduced by 8%, and the squalene content improved by 45% at 72 h, which demonstrated fluconazole's role in inhibiting squalene flow to cholesterol. As another typical UM with antioxidant capacity, the β-carotene production was increased by 53% at 96 h. The increase of squalene and β-carotene could boost intracellular oxidation resistance to protect fatty acids from oxidation. The NADPH was found to be 33% higher than that of the control at 96 h, which meant that the cells had more reducing power for fatty acid synthesis. Metabolic analysis further confirmed that regulation of sterols was closely related to glucose absorption, pigment biosynthesis and fatty acid production in Schizochytrium., Conclusion: This work first reported the effect of UM biosynthesis on fatty acid accumulation in Schizochytrium. The UM was found to affect fatty acid biosynthesis by changing cell membrane function, intracellular antioxidation and reducing power. We believe that this work provides valuable insights in improving PUFA and other valuable matters in microalgae.

The humpback grouper (Cromileptes altivelis) is a commercially valuable species of the family Epinephelidae; however, its marketization suffers from slow growth speed, low survival rate, and various pathogenic diseases. Lactococcus lactis and Schizochytrium limacinum are commonly used as immunostimulants due to their health benefits for the aquatic organisms. In the present study, we assessed the effects of dietary supplementation with L. lactis HNL12 combined with S. limacinum algal meal on the growth performances, innate immune response, and disease resistance of C. altivelis against Vibrio harveyi. The results showed that fish fed with a combination diet of L. lactis and S. limacinum exhibited significantly higher final weight, percent weight gain, and specific growth rate compared with groups fed with them alone. A bacterial challenge experiment indicated that the group fed with the L. lactis combined with S. limacinum diet achieved the highest relative percent of survival value (68.63%), suggesting that L. lactis and S. limacinum significantly improved the disease resistance against V. harveyi after a 4-week feeding trial. Moreover, the respiratory burst activity of macrophages of fish fed with a L. lactis combined with S. limacinum diet was significantly higher than that of fish fed the control diet after 1, 2, and 3 weeks of feeding. The serum superoxide dismutase of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to those fed the control diet after 1 and 2 weeks of feeding, while the serum alkaline phosphatase of fish fed with a L. lactis combined with S. limacinum diet after 2 and 4 weeks was significantly increased, compared to the control group. The serum lysozyme activities of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to the control group after 2 weeks of feeding. Furthermore, transcriptome sequencing of the C. altivelis head kidney was conducted to explore the immune-regulating effects of the L. lactis combined with S. limacinum diet on C. altivelis. A total of 86,919 unigenes, annotated by at least one of the reference databases (Nr, Swiss-Prot, GO, COG, and KEGG), were assembly yielded by de novo transcriptome. In addition, 157 putative differentially expressed genes (DEGs) were identified between the L. lactis combined with S. limacinum group and the control group. For pathway enrichment, the DEGs were categorized into nine KEGG pathways, which were mainly related to infective diseases, antigen processing and presentation, digestive system, and other immune system responses. The findings of this study suggest that the L. lactis combined with S. limacinum diet can induce positive effects on the growth, immunity, and disease resistance of C. altivelis against V. harveyi. This study expands our understanding of the synergistic combinations of probiotics and prebiotics in aquaculture., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The relevance of microalgae biotechnology for producing high-value compounds with biomedical application, such as polysaccharides, has been increasing. Despite this, the knowledge about the composition and structure of microalgae polysaccharides is still scarce. In this work, water-soluble polysaccharides from Nannochloropsis oculata were extracted, fractionated, structurally analysed, and subsequently tested in terms of immunostimulatory activity. A combination of sugar and methylation analysis with interaction data of carbohydrate-binding proteins using carbohydrate microarrays disclosed the complex structural features of the different polysaccharides. These analyses showed that the water-soluble polysaccharides fractions from N. oculata were rich in (β1→3, β1→4)-glucans, (α1→3)-, (α1→4)-mannans, and anionic sulphated heterorhamnans. The immunostimulatory assay highlighted that these fractions could also stimulate murine B-lymphocytes. Thus, the N. oculata water-soluble polysaccharides show potential to be further explored for immune-mediated biomedical applications., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Thraustochytrids are unicellular heterotrophic marine protists of the Stramenopile group, often considered as non-photosynthetic microalgae. They have been isolated from a wide range of habitats including deep sea, but are mostly present in waters rich in sediments and organic materials. They are abundant in mangrove forests where they are major colonizers, feeding on decaying leaves and initiating the mangrove food web. Discovered 80 years ago, they have recently attracted considerable attention due to their biotechnological potential. This interest arises from their fast growth, their specific lipid metabolism and the improvement of the genetic tools and transformation techniques. These organisms are particularly rich in ω3-docosahexaenoic acid (DHA), an 'essential' fatty acid poorly encountered in land plants and animals but required for human health. To produce their DHA, thraustochytrids use a sophisticated system different from the classical fatty acid synthase system. They are also a potential source of squalene and carotenoids. Here we review our current knowledge about the life cycle, ecophysiology, and metabolism of these organisms, with a particular focus on lipid dynamics. We describe the different pathways involved in lipid and fatty acid syntheses, emphasizing their specificity, and we report on the recent efforts aimed to engineer their lipid metabolism., (Copyright © 2019 Elsevier Ltd. All rights reserved.)