Chattonella marina blooms in a trophic gradient system: Interaction with environmental drivers.

For coastal eutrophication, lots of studies focused on the influence from environmental factors, especially nitrogen and phosphorus, on algae blooms. The interaction between algae and environmental factors has been often ignored. Using Chattonella marina, a dominant species in marine algal blooms, we established a trophic gradient system that simulated C. marina blooms at three trophic levels: eutrophic, mesotrophic, and oligotrophic, and examined the life history patterns of C. marina and the interactions with environmental factors. Increased trophic levels influenced the growth potential of C. marina, while its unique cyst reproduction allowed it to thrive in nutrient-limited environments. Adequate nutrients caused changes in dissolved oxygen (DO) and pH led by C. marina, with the carbonate system playing a crucial role in regulating pH under nutrient-limited conditions. Limiting the growth of C. marina in areas with low nutrient by manipulating reactive silicate (SiO32-) availability may prove effective. Nitrate (NO3-) was the preferred nutrient for C. marina when its concentration exceeded that of ammonium (NH4+). Phosphorus played a crucial role in the growth and proliferation of C. marina, especially when other nutrients were scarce. The findings of this study may provide valuable insights into the effective management and prevention of algae blooms.

Nitrogen and phosphorus stress as a tool to induce lipid production in microalgae.

Microalgae, capable of accumulating large amounts of lipids, are of great value for biodiesel production. The high cost of such production stimulates the search for cultivation conditions that ensure their highest productivity. Reducing the content of nitrogen and phosphorus in the culture medium is widely used to change the content and productivity of lipids in microalgae. Achieving the right balance between maximum growth and maximum lipid content and productivity is the primary goal of many experimental works to ensure cost-effective biodiesel production from microalgae. The content of nitrogen and phosphorus in nutrient media for algal cultivation after converted to nitrogen (-N) and phosphorus (-P) lies in an extensive range: from 0.007 g L- 1 to 0.417 g L- 1 and from 0.0003 g L- 1 to 0.227 g L- 1 and N:P ratio from 0.12:1 to 823.33:1. When studying nutritional stress in microalgae, no single approach is used to determine the experimental concentrations of nitrogen and phosphorus. This precludes the possibility of correct interpretation of the data and may lead to erroneous conclusions. This work results from the systematisation of information on using nitrogen and phosphorus restriction to increase the lipid productivity of microalgae of different taxonomic and ecological groups to identify future research directions. The results of 301 experiments were included in the analysis using the principal components method. The investigation considered various divisions and classes: Cyanobacteria, Rhodophyta, Dinophyta, Haptophyta, Cryptophyta, Heterokontophyta/Ochrophyta (Bacillariophyceae, Eustigmatophyceae, Xanthophyceae), Chlorophyta, and also the ratio N:P, the time of the experiment, the light intensity during cultivation. Based on the concentrations of nitrogen and phosphorus existing in various nutrient media, a general scheme for designating the supply of nutrient media for nitrogen (as NO3- or NH4+, N g L- 1) and phosphorus (as РO4-, P g L- 1) has been proposed: replete -N (˃0.4 g L- 1), moderate -N (0.4-0.2), moderate N-limitation (0.19-0.1), strong N-limitation (˂0.1), without nitrogen (0), replete -Р (˃0.2), moderate -P (0.2-0.02), moderate P-limitation (0.019-0.01), strong P-limitation (˂0.01), without phosphorus (0).

Orange Peel Waste as Feedstock for the Production of Glycerol-Free Biodiesel by the Microalgae Nannochloropsis oculata.

The bioconversion of agri-food waste into high-value products is gaining growing interest worldwide. Orange peel waste (OPW) is the main by-product of orange juice production and contains high levels of moisture and carbohydrates. In this study, the orange waste extract (OWE) obtained through acid hydrolysis of OPW was used as a substrate in the cultivation of the marine microalgae Nannochloropsis oculata. Photoheterotrophic (PH) and Photoautotrophic (PA) cultivations were performed in OWE medium and f/2 medium (obtained by supplementing OWE with macro- and micronutrients of f/2 medium), respectively, for 14 days. The biomass yields in PA and PH cultures were 390 mg L-1 and 450 mg L-1, while oil yields were 15% and 28%, respectively. The fatty acid (FA) profiles of PA cultures were mostly represented by saturated (43%) and monounsaturated (46%) FAs, whereas polyunsaturated FAs accounted for about 10% of the FAs. In PH cultures, FA profiles changed remarkably, with a strong increase in monounsaturated FAs (77.49%) and reduced levels of saturated (19.79%) and polyunsaturated (2.72%) FAs. Lipids obtained from PH cultures were simultaneously extracted and converted into glycerol-free biodiesel using an innovative microwave-assisted one-pot tandem protocol. FA methyl esters were then analyzed, and the absence of glycerol was confirmed. The FA profile was highly suitable for biodiesel production and the microwave-assisted one-pot tandem protocol was more effective than traditional extraction techniques. In conclusion, N. oculata used OWE photoheterotrophically, resulting in increased biomass and oil yield. Additionally, a more efficient procedure for simultaneous oil extraction and conversion into glycerol-free biodiesel is proposed.

Simultaneous DHA and organic selenium production by Schizochytrium sp.: a theoretical basis.

Docosahexaenoic acid (DHA) and selenium (Se) are nutrients that confer several health benefits to both humans and animals. Widespread use of DHA in milk powder and health products requires large-scale mass production via Schizochytrium sp., while Se intended for human consumption is produced as organic Se via yeast. However, producing these nutrients on an industrial scale is constrained by various factors. We found that supplementing Schizochytrium sp. with Na2SeO3 (0.5 mg/L) improves its biomass and DHA production and also provides organic Se. De novo assembled transcriptome and biochemical indicators showed that Na2SeO3 promotes forming acetyl coenzyme A and L-cysteine via the glycerol kinase and cysteine synthase pathways, promoting DHA synthesis through the polyketide synthase pathway. However, high doses of Na2SeO3 (5 mg/L) limited the biomass of Schizochytrium sp. and DHA content. This study provided a theoretical basis for the simultaneous production of organic Se and DHA via Schizochytrium sp.

Simultaneous production of high-value lipids in Schizochytrium sp. by synergism of chemical modulators.

The study focuses on the simultaneous improvement of biomass, lipid, and docosahexaenoic acid (DHA) productivities in a single reactor using modulator control strategies. The efficacy of three different biochemical modulators, sesamol (Ses), 6-benzylaminopurine (6-BAP), and ethylenediaminetetraacetic acid (EDTA), as potential stimulants in augmenting the biomass, lipid, and DHA production of Schizochytrium sp. MTCC 5890 was elucidated. After 48 h of cultivation, among tested modulators, the individual supplementation of 6-BAP and Ses showed improvement in biomass, lipid, and DHA accumulation by 28.2%, 56.1%, and 87.2% and 21.7%, 47.9%, and 91%, respectively, over the non-supplemented group. In addition, the cooperative effect of selected concentrations, i.e., 10 mgL-1 6-BAP and 200 mgL-1 Ses, further increased the productivities of biomass of 13.5 gL-1d-1 ± 0.66, lipid of 7.4 gL-1d-1 ± 0.69, and DHA of 3.2 gL-1d-1 ± 1.09 representing 8%, 39%, and 69% increase over the individual addition of 6-BAP or Ses, respectively, in batch culture. Supplementation with 6-BAP + Ses at 12 h of time point eventually increased the lipid yield to 15.6 ± 0.42 gL-1 from 7.88 ± 0.31 gL-1 (control) and DHA yield to 6.4 ± 0.11 gL-1 from 2.23 ± 0.09 gL-1 (control), respectively. Furthermore, the process was optimized in continuous culture supplemented with 6-BAP + Ses for enhanced productivities. Continuous culture resulted in maximum biomass (2.04 ± 1.12 gL-1 day-1), lipid (1.0 ± 0.73 gL-1 day-1), and DHA (0.386 ± 0.22 gL-1 day-1) productivities, which were higher as compared with the batch and fed-batch processes by 26 ± 1.21%, 22 ± 1.01%, and 21 ± 0.98% and 24 ± 0.45%, 16 ± 0.38%, and 14 ± 0.12%, respectively. This work represents the potential application of the combined effect of modulators for the simultaneous enhancement of biomass production and lipid and DHA productivities. KEY POINTS: • The cumulative study of 6-BAP and sesamol proved to be more efficient in the simultaneous production of biomass, lipid, and DHA in a single reactor. • Addition of a combination of 6-BAP + Ses remarkably increased the biomass, lipid, and DHA productivities in tandem in continuous culture.

Bioprocess conditions and regulation factors to optimize squalene production in thraustochytrids.

Squalene is a widely distributed natural triterpene, as it is a key precursor in the biosynthesis of all sterols. It is a compound of high commercial value worldwide because it has nutritional, medicinal, pharmaceutical, and cosmetic applications, due to its different biological properties. The main source of extraction has been shark liver oil, which is currently unviable on a larger scale due to the impacts of overexploitation. Secondary sources are mainly vegetable oils, although a limited one, as they allow low productive yields. Due to the diversity of applications that squalene presents and its growing demand, there is an increasing interest in identifying sustainable sources of extraction. Wild species of thraustochytrids, which are heterotrophic protists, have been identified to have the highest squalene content compared to bacteria, yeasts, microalgae, and vegetable sources. Several studies have been carried out to identify the bioprocess conditions and regulation factors, such as the use of eustressors that promote an increase in the production of this triterpene; however, studies focused on optimizing their productive yields are still in its infancy. This review includes the current trends that also comprises the advances in genetic regulations in these microorganisms, with a view to identify the culture conditions that have been favorable in increasing the production of squalene, and the influences that both bioprocess conditions and applied regulation factors partake at a metabolic level.

Effects of sodium selenite on the growth, biochemical composition and selenium biotransformation of the filamentous microalga Tribonema minus.

This study aimed to investigate the physiological and biochemical responses of filamentous microalga Tribonema minus to different Na2SeO3 concentrations and its selenium absorption and metabolism to evaluate the potential in treating selenium-containing wastewater. The results showed that low Na2SeO3 concentrations promoted growth by increasing chlorophyll content and antioxidant capacity, whereas high concentrations caused oxidative damage. Although Na2SeO3 exposure reduced lipid accumulation compared with the control, it significantly increased carbohydrate, soluble sugar, and protein contents, with the highest carbohydrate productivity of 117.97 mg/L/d at 0.5 mg/L Na2SeO3. Furthermore, this alga effectively absorbed Na2SeO3 in the growth medium and converted most of it into volatile selenium and a small part into organic selenium (predominantly as selenocysteine), showing strong selenite removal efficacy. This is the first report on the potential of T. minus to produce valuable biomass while removing selenite, providing new insights into the economic feasibility of bioremediation of selenium-containing wastewater.

Functional metabolism pathways of significantly regulated genes in Nannochloropsis oceanica with various nitrogen/phosphorus nutrients for CO2 fixation.

To determine the optimal CO2 concentration for microalgal biomass cultivated with industrial flue gas and improve carbon fixation capacity and biomass production. Functional metabolism pathways of significantly regulated genes in Nannochloropsis oceanica (N. oceanica) with various nitrogen/phosphorus (N/P) nutrients for CO2 fixation were comprehensively clarified. At 100 % N/P nutrients, the optimum CO2 concentration was 70 % and the maximum biomass production of microalgae was 1.57 g/L. The optimum CO2 concentration was 50 % for N or P deficiency and 30 % for both N and P deficiency. The optimal combination of CO2 concentration and N/P nutrients caused significant up regulation of proteins related to photosynthesis and cellular respiration in the microalgae, enhancing photosynthetic electron transfer efficiency and carbon metabolism. Microalgal cells with P deficiency and optimal CO2 concentration expressed many phosphate transporter proteins to enhance P metabolism and N metabolism to maintain a high carbon fixation capacity. However, inappropriate combination of N/P nutrients and CO2 concentrations caused more errors in DNA replication and protein synthesis, generating more lysosomes and phagosomes. This inhibited carbon fixation and biomass production in the microalgae with increased cell apoptosis.

Protist-Lactic Acid Bacteria Co-Culture as a Strategy to Bioaccumulate Polyunsaturated Fatty Acids in the Protist Aurantiochytrium sp. T66.

Thraustochytrids are aquatic unicellular protists organisms that represent an important reservoir of a wide range of bioactive compounds, such as essential polyunsaturated fatty acids (PUFAs) such as arachidonic acid (ARA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), which are involved in the regulation of the immune system. In this study, we explore the use of co-cultures of Aurantiochytrium sp. and bacteria as a biotechnological tool capable of stimulating PUFA bioaccumulation. In particular, the co-culture of lactic acid bacteria and the protist Aurantiochytrium sp. T66 induce PUFA bioaccumulation, and the lipid profile was evaluated in cultures at different inoculation times, with two different strains of lactic acid bacteria capable of producing the tryptophan dependent auxins, and one strain of Azospirillum sp., as a reference for auxin production. Our results showed that the Lentilactobacillus kefiri K6.10 strain inoculated at 72 h gives the best PUFA content (30.89 mg g-1 biomass) measured at 144 h of culture, three times higher than the control (8.87 mg g-1 biomass). Co-culture can lead to the generation of complex biomasses with higher added value for developing aquafeed supplements.

Expression of glycerol-3-phosphate acyltransferase increases non-polar lipid accumulation in Nannochloropsis oceanica.

Microalgae are considered a suitable production platform for high-value lipids and oleochemicals. Several species including Nannochloropsis oceanica produce large amounts of essential [Formula: see text]-3 polyunsaturated fatty acids (PUFAs) which are integral components of food and feed and have been associated with health-promoting effects. N. oceanica can further accumulate high contents of non-polar lipids with chemical properties that render them a potential replacement for plant oils such as palm oil. However, biomass and lipid productivities obtained with microalgae need to be improved to reach commercial feasibility. Genetic engineering can improve biomass and lipid productivities, for instance by increasing carbon flux to lipids. Here, we report the overexpression of glycerol-3-phosphate acyltransferase (GPAT) in N. oceanica during favorable growth conditions as a strategy to increase non-polar lipid content. Transformants overproducing either an endogenous (NoGPAT) or a heterologous (Acutodesmus obliquus GPAT) GPAT enzyme targeted to the endoplasmic reticulum had up to 42% and 51% increased non-polar lipid contents, respectively, compared to the wild type. Biomass productivities of transformant strains were not substantially impaired, resulting in lipid productivities that were increased by up to 37% and 42% for NoGPAT and AoGPAT transformants, respectively. When exposed to nutrient stress, transformants and wild type had similar lipid contents, suggesting that GPAT enzyme exerts strong flux control on lipid synthesis in N. oceanica under favorable growth conditions. NoGPAT transformants further accumulated PUFAs in non-polar lipids, reaching a total of 6.8% PUFAs per biomass, an increase of 24% relative to the wild type. Overall, our results indicate that GPAT is an interesting target for engineering of lipid metabolism in microalgae, in order to improve non-polar lipid and PUFAs accumulation in microalgae.

Enhanced production of high-value polyunsaturated fatty acids (PUFAs) from potential thraustochytrid Aurantiochytrium sp.

Due to growing health concerns, the urban population is utterly inclined towards a healthy lifestyle and incorporated nutritional food supplements to lower common health risks. The ω-3 and ω-6 PUFAs consumption is increasing, hence alternative commercial production is essentially developed. The microbial source is an emerging platform to overcome the global demand for omega PUFAs. Marine oleaginous protist Aurantiochytrium sp. found a potential source to produce substantial DHA and SFA. The objective of the present research was to enhance the PUFA yield by optimizing maximum tolerable glucose concentration with a suitable nitrogen ratio (10:1). The maximum lipid and DHA yield and content were determined 4.30, 1.34 g/L, and 62.4, 33.49 % of total biomass and lipid at 30 g/L glucose respectively, which is one of among highest reported, however relative PUFA was maximum 46.97 % (DHA) in total lipid at 10 g/L glucose. Remaining 42-53.6 % SFA could be used for biodiesel.

Role of key enzymes in the production of docosahexaenoic acid (DHA) by Thraustochytrium sp. T01.

Docosahexaenoic acid (DHA) is an essential dietary supplement that is highly coveted due to its benefits for human health. Extensive research has been conducted for the sustainable commercial production of DHA by various strains in thraustochytrid family due to the accumulation of higher lipid content in the cells. The current study is focused on improving DHA production by investigating various key enzymes like glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME), and ATP-citrate lyase (ACL) involved in DHA production using Thraustochytrium sp. T01. The growth of this strain was compared in batch and fed-batch mode. The fed-batch yielded better Dry cell weight (40 g L-1), lipid (27.75 g L-1 or 693 mg g-1 of DCW), and DHA contents (11.10 g L-1 or 277 mg g-1 of DCW). G6PDH activity increased 4-fold during the glucose fed-batch, but ME and ACL did not increase significantly. Furthermore, a study was conducted to determine the effects of organic acids (pyruvate and malate) on key enzyme activities. The addition of pyruvate increased the lipid content by 1.35-fold, and ACL activity by 10-fold as compared with control (without added organic acids). Malate addition into the culture media increased DHA content 1.4-fold, and ME activity increased 14-fold compared with control.

Effect of phosphorus limitation on Se uptake efficiency in the microalga Nannochloropsis oceanica.

Microalgae are considered an efficient accumulator and promising source of Se for feed additive purposes. This study aimed at investigating, for the first time, the effect of phosphorus limitation on Se accumulation and uptake efficiency in N.oceanica. A range of phosphorus concentrations (0-2470 µM) were tested in either the presence or absence of sodium selenite (0, 5, 30 µM). Se accumulation was increased up to 16-fold and Se uptake efficiency was increased up to 3.6-fold under phosphorus growth-limiting concentrations. N.oceanica was then cultivated in a 1.8L flat-panel photobioreactor in batch operation under two phosphorus growth-limiting concentrations (250 and 750 µM) where the accumulation of Se in the microalgal biomass, as well as its presence in the spent medium were analysed. This study is the first to investigate the effect of phosphorus limitation for increasing Se accumulation in microalgae, and to prevent the release of Se in wastewater.

Microalgae oil from Schizochytrium sp. alleviates obesity and modulates gut microbiota in high-fat diet-fed mice.

Omega-3 PUFAs rich in fish oil are believed to prevent obesity by improving lipid metabolism and regulating gut microbiota. Microalgae oil is considered as an alternative source of omega-3 PUFAs owing to diminishing fish resources. Schizochytrium microalgae oil (SMO), with a high DHA proportion, is a promising source for commercial DHA production. However, its weight-loss and gut microbiota-regulating properties are not well studied. Here we compared the obesity reducing effects of SMO, commercial fish oil (FO) and a weight-loss drug, Orlistat (OL), in a high-fat diet (HFD) induced obesity mouse model. We found that SMO is comparable to commercial FO and OL with regard to weight loss, and it even exhibits the weight-loss effects earlier than FO and OL. It can efficiently inhibit the expression of lipogenesis-related genes and induce the expression of lipolysis-related genes. Moreover, SMO has different gut microbiota modulating effects from those of FO and OL. It does not influence the diversity of bacterial community, but does increase the abundance of several beneficial SCFAs-producing bacteria and inhibits obesity-promoting Desulfovibrio and several pathogens. We also found that SMO recovers the HFD-disturbed metabolic capability of gut microbiota. It can increase the abundance of several metabolism-related pathways, such as those of amino acids, SCFAs and bile acid, and decrease the level of the LPS biosynthesis pathway, which probably contributes to an improvement of lipid metabolism and restoration of the colonic mucosal barrier impaired by HFD. Our data suggest that SMO can be used as a superior dietary supplement for alleviating obesity.

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.

Enhancement of Squalene Production by Constitutive Expression of the 3-Hydroxy-3-Methylglutaryl-CoA Reductase in Aurantiochytrium sp. 18W-13a.

Squalene has a wide range of applications in the industry sectors of dietary supplements, cosmetics, immunization, and pharmaceuticals. Yet, suitable organisms as the source of squalene are limited. It is reported that the thraustochytrid Aurantiochytrium sp. strain 18W-13a can accumulate high content of squalene. However, squalene production in this organism is fluctuated under various conditions and is not yet optimized for commercialization. In this organism, the mevalonate pathway supplies isopentenyl pyrophosphate, the initial substrate for squalene production. In this pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the rate-limiting enzyme. We found that the HMGR activity had a strong positive correlation with the squalene contents in the strain. We constitutively expressed the HMGR in this organism and found that the transformant showed increased and stable production of squalene as well as carotenoids and biomass. These results clearly indicated that the HMGR expression is the bottleneck of squalene synthesis in Aurantiochytrium sp.

Producing high value unsaturated fatty acid by whole-cell catalysis using microalga: A case study with Tribonema minus.

High value unsaturated fatty acids can be produced by de novo synthesis in microalgal cells, especially via heterotrophic cultivation. Unfortunately, the lipid accumulation of heterotrophic microalgae cannot be improved efficiently in conventional ways. Here we reported heterotrophic Tribonema minus, a promising resource for the production of palmitoleic acid which has increasing demands in health service for patients with metabolic syndrome, as whole-cell biocatalyst to develop a novel way of shifting low value exogenous saturated fatty acids to high value ones. Results showed that myristic acid is the best precursor for whole-cell catalysis; it elevated the lipid content of T. minus to 42.2%, the highest among the tried precursors. The influences of cultivation condition on the utilization of extrinsic myristic acid and lipid accumulation were also determined. Under the optimized condition, the lipid content reached as high as 48.9%. In addition, our findings showed that ~13.0% of C16:1 in T. minus is derived from extrinsic myristic acid, and 30.1% of metabolized precursor is converted into heterologous fatty acids. Thus, a feasible approach for both increasing the value of low value saturated fatty acid by bioconversion and enhancing the lipid accumulation in microalgae is proposed by supplementing extrinsic myristic acid.

Astaxanthin and eicosapentaenoic acid production by S4, a new mutant strain of Nannochloropsis gaditana.

BACKGROUND: Astaxanthin is a ketocarotenoid with high antioxidant power used in different fields as healthcare, food/feed supplementation and as pigmenting agent in aquaculture. Primary producers of astaxanthin are some species of microalgae, unicellular photosynthetic organisms, as Haematococcus lacustris. Astaxanthin production by cultivation of Haematococcus lacustris is costly due to low biomass productivity, high risk of contamination and the requirement of downstream extraction processes, causing an extremely high price on the market. Some microalgae species are also primary producers of omega-3 fatty acids, essential nutrients for humans, being related to cardiovascular wellness, and required for visual and cognitive development. One of the main well-known producers of omega-3 fatty eicosapentaenoic acid (EPA) is the marine microalga Nannochloropsis gaditana (named also Microchloropsis gaditana): this species has been already approved by the Food and Drug Administration (FDA) for human consumption and it is characterized by a fast grow phenotype. RESULTS: Here we obtained by chemical mutagenesis a Nannochloropsis gaditana mutant strain, called S4, characterized by increased carotenoid to chlorophyll ratio. S4 strain showed improved photosynthetic activity, increased lipid productivity and increased ketocarotenoids accumulation, producing not only canthaxanthin but also astaxanthin, usually found only in traces in the WT strain. Ketocarotenoids produced in S4 strain were extractible in different organic solvents, with the highest efficiency observed upon microwaves pre-treatment followed by methanol extraction. By cultivation of S4 strain at different irradiances it was possible to produce up to 1.3 and 5.2 mgL-1 day-1 of ketocarotenoids and EPA respectively, in a single cultivation phase, even in absence of stressing conditions. Genome sequencing of S4 strain allowed to identify 199 single nucleotide polymorphisms (SNP): among the mutated genes, mutations in a carotenoid oxygenase gene and in a glutamate synthase gene could explain the different carotenoids content and the lower chlorophylls content, respectively. CONCLUSIONS: By chemical mutagenesis and selection of strain with increased carotenoids to chlorophyll ratio it was possible to isolate a new Nannochloropsis gaditana strain, called S4 strain, characterized by increased lipids and ketocarotenoids accumulation. S4 strain can thus be considered as novel platform for ketocarotenoids and EPA production for different industrial applications.

Osteogenic activities of four calcium-chelating microalgae peptides.

BACKGROUND: Adequate calcium intake is necessary to prevent osteoporosis, which poses significant public health challenges. The natural bioactive peptide calcium chelates have been regarded as superior calcium supplements. Microalgae peptides are regarded as potential candidates for protection from bone loss in osteoporosis. This study aimed to prepare microalgae calcium-chelating peptides from four microalgae proteins and assess their osteogenic activities in osteoporosis-like zebrafish. RESULTS: After in vitro gastrointestinal digestion, 4.42% Chlorella pyrenoidosa protein, 2.74% Nannochloropsis oceanica protein, 6.07% Arthospira platensis protein and 10.47% Dunaliella salina protein were retained. The calcium-chelating capacities of four microalgae protein hydrolysates (MPHs) ranged from 14.10 ± 7.16% to 34.11 ± 9.34%. CaCl2 addition increased the maximum absorption peaks, absorption intensities and particle sizes of MPHs. Calcium-chelating MPHs showed stronger osteogenic activities than MPHs in the osteoporosis-like zebrafish model, with significantly increased mineralized tissue area and integrated optical density. CONCLUSION: Microalgae proteins have favorable digestibilities. Among the four MPHs, Nannochloropsis oceanica protein hydrolysates showed the highest calcium-chelating capacity, which might be due to its high degree of hydrolysis after in vitro digestion and high content of Ser, Tyr, Thr, Asp and Glu. The absorption intensities and particle sizes of MPHs both increased after calcium addition. MPH treatment could reverse dexamethasone-induced osteoporosis of zebrafish, and MPHs-Ca chelates showed higher osteogenic activities in osteoporosis-like phenotype zebrafish. © 2022 Society of Chemical Industry.

Media Supplementation with Mannitol and Biotin Enhances Squalene Production of Thraustochytrium ATCC 26185 through Increased Glucose Uptake and Antioxidative Mechanisms.

Media supplementation with exogenous chemicals is known to stimulate the accumulation of important lipids produced by microalgae and thraustochytrids. However, the roles of exogenous chemicals in promoting and preserving the terpenoids pool of thraustochytrids have been rarely investigated. Here, we realized the effects of two media supplements-mannitol and biotin-on the biomass and squalene production by a thraustochytrid strain (Thraustochytrium sp. ATCC 26185) and elucidated their mechanism of action. A significant change in the biomass was not evident with the exogenous addition of these supplements. However, with mannitol (1 g/L) supplementation, the ATCC 26185 culture achieved the best concentration (642 ± 13.6 mg/L) and yield (72.9 ± 9.6 mg/g) of squalene, which were 1.5-fold that of the control culture (non-supplemented). Similarly, with biotin supplementation (0.15 mg/L), the culture showed 459 ± 2.9 g/L and 55.7 ± 3.2 mg/g of squalene concentration and yield, respectively. The glucose uptake rate at 24 h of fermentation increased markedly with mannitol (0.31 g/Lh-1) or biotin (0.26 g/Lh-1) supplemented culture compared with non-supplemented culture (0.09 g/Lh-1). In addition, the reactive oxygen species (ROS) level of culture supplemented with mannitol remained alleviated during the entire period of fermentation while it alleviated after 24 h with biotin supplementation. The ∆ROS with mannitol was better compared with biotin supplementation. The total antioxidant capacity (T-AOC) of the supplemented culture was more than 50% during the late stage (72-96 h) of fermentation. Our study provides the potential of mannitol and biotin to enhance squalene yield and the first lines of experimental evidence for their protective role against oxidative stress during the culture of thraustochytrids.