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Crypthecodinium cohnii is a marine microalga that can accumulate high amounts of polyunsaturated fatty acids (PUFAs) and thus replace conventional routes of fish oil production. They are associated with the destruction of marine resources and multiple downstream/purification complications. The major drawbacks of using C. cohnii for industrial-scale production are associated with low PUFA productivity. One of the means of increasing the PUFA synthesis rate is to maintain the medium component concentrations at optimal values throughout cultivation, thus increasing PUFA production efficiency, which can result in the successful transfer of the process to pilot and/or industrial scale. The goal of the present research was to develop techniques for increasing the efficiency of PUFA production via C. cohnii cultivation. Multiple experiments were carried out to test and fine-tune the cultivation medium composition and oxygen transfer factors. The biomass yields from individual components, yeast extract, sea salts, and glucose amounted to 5.5, 0.65, and 0.61 g·g−1, respectively. C. cohnii cell susceptibility to mechanical damage was experimentally evaluated. Power inputs of <276.5 W/m3 did not seem to promote cell destruction when Pitched-blade impellers were used. The obtained cultivation conditions were shown to be efficient in terms of increasing the biomass productivity and the omega-3 fatty acid content in C. cohnii. By using the applied methods, the maximal biomass productivity reached 8.0 g·L−1·day−1, while the highest obtained biomass concentration reached 110 g·L−1. A steady increase in the concentration of PUFAs during cultivation was observed from the FTIR data. [ABSTRACT FROM AUTHOR]

Crypthecodinium cohnii is a marine microalga that can accumulate high amounts of polyunsaturated fatty acids (PUFAs) and thus replace conventional routes of fish oil production. They are associated with the destruction of marine resources and multiple downstream/purification complications. The major drawbacks of using C. cohnii for industrial-scale production are associated with low PUFA productivity. One of the means of increasing the PUFA synthesis rate is to maintain the medium component concentrations at optimal values throughout cultivation, thus increasing PUFA production efficiency, which can result in the successful transfer of the process to pilot and/or industrial scale. The goal of the present research was to develop techniques for increasing the efficiency of PUFA production via C. cohnii cultivation. Multiple experiments were carried out to test and fine-tune the cultivation medium composition and oxygen transfer factors. The biomass yields from individual components, yeast extract, sea salts, and glucose amounted to 5.5, 0.65, and 0.61 g·g−1, respectively. C. cohnii cell susceptibility to mechanical damage was experimentally evaluated. Power inputs of 3 did not seem to promote cell destruction when Pitched-blade impellers were used. The obtained cultivation conditions were shown to be efficient in terms of increasing the biomass productivity and the omega-3 fatty acid content in C. cohnii. By using the applied methods, the maximal biomass productivity reached 8.0 g·L−1·day−1, while the highest obtained biomass concentration reached 110 g·L−1. A steady increase in the concentration of PUFAs during cultivation was observed from the FTIR data.

二十二碳六烯酸（DHA）是一种ω-3 系多不饱和脂肪酸，对人体生长发育和健康具有重要作用，但其传统来源并不稳定且会引入鱼腥味，因此积极开发DHA新来源具有重要的研究价值和市场潜力。介绍了DHA的结构、理化性质、功能特性和来源，并从微生物产DHA途径，高产DHA藻种裂壶藻和寇氏隐甲藻的特点、诱变选育种情况、摇瓶培养条件和发酵工艺优化等方面阐述了微生物发酵生产DHA的研究情况。另外，从食用油和乳液两个方面对DHA藻油在食品中的应用情况进行了介绍。目前以裂壶藻和寇氏隐甲藻发酵生产DHA已取得初步成功，可以为现有生产提供理论基础，但有关胞内合成DHA和精准调控实现DHA增产还有待进一步研究。 Docosahexaenoic acid (DHA) is a kind of ω-3 polyunsaturated fatty acids, playing an important role in human physical development and health. However, the traditional sources for DHA are unstable and introduce the fishy smell for the products. Therefore, developing new sources for DHA actively shows important research value and market potential. The structure, physicochemical properties, functional properties and sources of DHA were introduced. The research on the production of DHA by microbial fermentation was described in terms of synthesis pathway of DHA, the characteristics, the mutagenesis breeding situation, the optimization of shake flask culture conditions and fermentation process of the high DHA-producing algae strains Schiztochytrium and Crypthecodinium cohnii. In addition, the application of DHA algal oil was introduced from two aspects of edible oils and emulsions. At present, the production of DHA by fermentation with Schizochytrium and Crypthecodinium cohnii has achieved initial success, which can provide a theoretical basis for the production. However, the intracellular synthesis of DHA and the precise regulation of DHA production need further research.

Chrypthecodinium cohnii lipids have been almost exclusively used as a source of Docosahexaenoic acid (DHA). Such an approach wastes the remaining microalgal lipid fraction. The present work presents a novel process to produce C. cohnii biomass, using low-cost industrial by-products (raw glycerol and corn steep liquor), in a 7L-bioreactor, under fed-batch regime. At the end of the fermentation, the biomass concentration reached 9.2 g/L and the lipid content and lipid average productivity attained 28.0% (w/w dry cell weight) and 13.6 mg/L h, respectively. Afterwards the microalgal biomass underwent a saponification reaction to produce fatty acid (FA) soaps, which were further converted into FA ethyl ester (FA EE). C. cohnii FA EE mixture was then fractionated, using the urea complexation method at different temperatures, in order to obtain a polyunsaturated fatty acid ethyl ester (PUFA EE) rich fraction, that could be used for food/pharmaceutical/cosmetic purposes, and a saturated fatty acid ethyl ester (SAT EE) rich fraction, which could be used as biodiesel. The temperature that promoted the best separation between PUFA and SAT EE, was −18 °C, resulting in a liquid fraction with 91.6% (w/w) DHA, and a solid phase with 88.2% of SAT and monounsaturated fatty acid ethyl ester (MONOUNSAT), which could be used for biodiesel purposes after a hydrogenation step. [ABSTRACT FROM AUTHOR]

Recovery of omega-3 fatty acids (FAs) from microalgae constitutes a valuable alternative to fish oil, and offers the ability to isolate a single ω-3 FA, instead of a mixture. In the framework of the concept of circular economy and sustainability, in the current work, an alternative carbon source was used as feed for oleaginous microalga Crypthecodinium cohnii cultivation and, consequently, production of the omega-3 FA, docosahexaenoic acid (DHA). The conditions that improve DHA production in a fed-batch culture of C. cohnii, using an organic acid-rich liquid fraction of dark fermented biowaste as feed, were studied and optimized. The process was mathematically approached focusing on a proper Design of Experiments (DoE) method coupled with Response Surface Methodology (RSM). The temperature of 23 °C and the use of yeast extract (YE) as initial nitrogen source favored the amount of DHA recovered. In addition, the increase of the volatile FA (VFA) concentration of the feed enhanced the recovered cell biomass. In order to propose a final fermentation protocol with industrial potential and evaluate the annual total cost for the production of DHA, fermentation process was scaled-up and economically evaluated through cost analysis. The increase of bioreactor size from 1 to 20 m3 leads to a significant decrease in the cost of produced DHA while leveling at higher capacities.

Docosahexaenoic acid (DHA, 22:6n-3) plays significant roles in enhancing human health and preventing human diseases. The heterotrophic marine dinoflagellate Crypthecodinium cohnii is a good candidate to produce high-quality DHA. To overcome the inhibition caused by the fermentation supernatant in the late fermentation stage of DHA-producing C. cohnii , fermentation supernatant-based adaptive laboratory evolution (FS-ALE) was conducted. The cell growth and DHA productivity of the evolved strain (FS280) obtained after 280 adaptive cycles corresponding to 840 days of evolution were increased by 161.87 and 311.23%, respectively, at 72 h under stress conditions and increased by 19.87 and 51.79% without any stress compared with the starting strain, demonstrating the effectiveness of FS-ALE. In addition, a comparative proteomic analysis identified 11,106 proteins and 910 differentially expressed proteins, including six stress-responsive proteins, as well as the up- and downregulated pathways in FS280 that might contribute to its improved cell growth and DHA accumulation. Our study demonstrated that FS-ALE could be a valuable solution to relieve the inhibition of the fermentation supernatant at the late stage of normal fermentation of heterotrophic microalgae. [ABSTRACT FROM AUTHOR]

Docosahexaenoic acid (DHA, 22:6n-3) plays significant roles in enhancing human health and preventing human diseases. The heterotrophic marine dinoflagellate Crypthecodinium cohnii is a good candidate to produce high-quality DHA. To overcome the inhibition caused by the fermentation supernatant in the late fermentation stage of DHA-producing C. cohnii, fermentation supernatant-based adaptive laboratory evolution (FS-ALE) was conducted. The cell growth and DHA productivity of the evolved strain (FS280) obtained after 280 adaptive cycles corresponding to 840 days of evolution were increased by 161.87 and 311.23%, respectively, at 72 h under stress conditions and increased by 19.87 and 51.79% without any stress compared with the starting strain, demonstrating the effectiveness of FS-ALE. In addition, a comparative proteomic analysis identified 11,106 proteins and 910 differentially expressed proteins, including six stress-responsive proteins, as well as the up- and downregulated pathways in FS280 that might contribute to its improved cell growth and DHA accumulation. Our study demonstrated that FS-ALE could be a valuable solution to relieve the inhibition of the fermentation supernatant at the late stage of normal fermentation of heterotrophic microalgae.

Abstract Background Docosahexaenoic acid (DHA) is essential for human diet. However, high production cost of DHA using C. cohnii makes it currently less competitive commercially, which is mainly caused by low DHA productivity. In recent years, repeated fed-batch strategies have been evaluated for increasing the production of many fermentation products. The reduction in terms of stability of culture system was one of the major challenges for repeated fed-batch fermentation. However, the possible mechanisms responsible for the decreased stability of the culture system in the repeated fed-batch fermentation are so far less investigated, restricting the efforts to further improve the productivity. In this study, a repeated fed-batch strategy for DHA production using C. cohnii M-1-2 was evaluated to improve DHA productivity and reduce production cost, and then the underlying mechanisms related to the gradually decreased stability of the culture system in repeated fed-batch culture were explored through LC– and GC–MS metabolomic analyses. Results It was discovered that glucose concentration at 15–27 g/L and 80% medium replacement ratio were suitable for the growth of C. cohnii M-1-2 during the repeated fed-batch culture. A four-cycle repeated fed-batch culture was successfully developed and assessed at the optimum cultivation parameters, resulting in increasing the total DHA productivity by 26.28% compared with the highest DHA productivity of 57.08 mg/L/h reported using C. cohnii, including the time required for preparing seed culture and fermentor. In addition, LC– and GC–MS metabolomics analyses showed that the gradually decreased nitrogen utilization capacity, and down-regulated glycolysis and TCA cycle were correlated with the decreased stability of the culture system during the long-time repeated fed-batch culture. At last, some biomarkers, such as Pyr, Cit, OXA, FUM, l-tryptophan, l-threonine, l-leucine, serotonin, and 4-guanidinobutyric acid, correlated with the stability of culture system of C. cohnii M-1-2 were identified. Conclusions The study proved that repeated fed-batch cultivation was an efficient and energy-saving strategy for industrial production of DHA using C. cohnii, which could also be useful for cultivation of other microbes to improve productivity and reduce production cost. In addition, the mechanisms study at metabolite level can also be useful to further optimize production processes for C. cohnii and other microbes.

Abstract Docosahexaenoic acid (DHA) enriched in brain can yield many important degradation products after the attack of hydroxyl radicals, which is known to serve as a nutraceutical and neuroprotective effects. Oxidative stress is a commonly observed feature of Alzheimer’s disease (AD). Therefore, uniformly radiolabeled DHA plays an important role in studying the oxidative fate of DHA in vivo and vitro. However, carbon isotope labeled DHA isn’t commercially available now. The heterotrophic microalgae Crypthecodinium cohnii (C. cohnii) has been identified as a prolific producer of DHA. In this study, the growth rate and DHA production in C. cohnii were optimized in a new defined media, and the biosynthesis of U-13C-DHA from U-13C-glucose and U-14C-DHA from U-14C-glucose were analyzed by HPLC–MS/MS. Approximately 40 nmoles of U-13C-DHA with higher isotopic purity of 96.8% was produced in a 300 μL batch, and ~ 0.23 μCi of U-14C-DHA with significant specific activity of 5–6 Ci/mol was produced in a 300 μL batch. It was found that C. cohnii had the optimal growth and DHA accumulation at 25 °C in this defined media (C/N = 10). An efficient protocol for the biosynthesis of U-13C-DHA and U-14C-DHA were set up firstly, which provides the basic support for the analysis of oxidative degradation products of DHA in AD.

• Immobilized Z. mobilis converted sucrose to ethanol that was consumed by C. cohnii. • Levansucrase (sacB) and ndh -negative double mutant Zm6- ndh-sacB was constructed. • Immobilized Zm6- ndh-sacB converted sucrose to ethanol aerobically with a high yield. • Fed-batch with Zm6- ndh-sacB fermentation medium and cocultivations were carried out. • Syntrophy with immobilized Zm6- ndh-sacB yielded C. cohnii biomass with up to 3% DHA. Marine heterotrophic dinoflagellate Crypthecodinium cohnii is an aerobic oleaginous microorganism that accumulates intracellular lipid with high content of 4,7,10,13,16,19-docosahexaenoic acid (DHA), a polyunsaturated ω−3 (22:6) fatty acid with multiple health benefits. C. cohnii can grow on glucose and ethanol, but not on sucrose or fructose. For conversion of sucrose-containing renewables to C. cohnii DHA, we investigated a syntrophic process, involving immobilized cells of ethanologenic bacterium Zymomonas mobilis for fermenting sucrose to ethanol. The non-respiring, NADH dehydrogenase-deficient Z. mobilis strain Zm6- ndh , with high ethanol yield both under anaerobic and aerobic conditions, was taken as the genetic background for inactivation of levansucrase (sacB). SacB mutation eliminated the levan-forming activity on sucrose. The double mutant Zm6- ndh - sacB cells were immobilized in Ca alginate, and applied for syntrophic conversion of sucrose to DHA of C. cohnii , either taking the ethanol-containing fermentation medium from the immobilized Z. mobilis for feeding to the C. cohnii fed-batch culture, or directly coculturing the immobilized Zm6- ndh - sacB with C. cohnii on sucrose. Both modes of cultivation produced C. cohnii CCMP 316 biomass with DHA content around 2–3 % of cell dry weight, corresponding to previously reported results for this strain on glucose. [ABSTRACT FROM AUTHOR]

Cultivation conditions especially agitation and aeration have critical roles on Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) formation. In this study, for the first time, different air bubble sizes were considered, in a 5-L bubble column bioreactor; and the effect of bubble size on growth and on DHA production by dinoflagellate microalgae C. cohnii was investigated. The cultivation was carried out under 0.5 vvm aeration flow rate, 28 °C, and pH ± 7. The highest yield of DHA (1.4 g DHA L−1) was achieved for 0.36-cm air bubble diameter; the corresponding biomass amount was 17.6 g. The suitable conditions during the logarithmic stage of growth were provided for 0.36-cm air bubble diameter, while oxygen limitation was observed in the culture medium afterwards. The concentration of the microalgal DHA was increased in oxygen limitation conditions. Our results indicate that the air bubble diameter is the most influencing factor on DHA production in a bubble column bioreactor. Current research indicates that optimum bubble size in bubble column bioreactor can lead to optimal conditions for growth and DHA production like two-stage oxygen supply feeding strategy. [ABSTRACT FROM AUTHOR]

Chrypthecodinium cohnii lipids have been almost exclusively used as a source of Docosahexaenoic acid (DHA). Such an approach wastes the remaining microalgal lipid fraction. The present work presents a novel process to produce C. cohnii biomass, using low-cost industrial by-products (raw glycerol and corn steep liquor), in a 7L-bioreactor, under fed-batch regime. At the end of the fermentation, the biomass concentration reached 9.2 g/L and the lipid content and lipid average productivity attained 28.0% (w/w dry cell weight) and 13.6 mg/L h, respectively. Afterwards the microalgal biomass underwent a saponification reaction to produce fatty acid (FA) soaps, which were further converted into FA ethyl ester (FA EE). C. cohnii FA EE mixture was then fractionated, using the urea complexation method at different temperatures, in order to obtain a polyunsaturated fatty acid ethyl ester (PUFA EE) rich fraction, that could be used for food/pharmaceutical/cosmetic purposes, and a saturated fatty acid ethyl ester (SAT EE) rich fraction, which could be used as biodiesel. The temperature that promoted the best separation between PUFA and SAT EE, was −18 °C, resulting in a liquid fraction with 91.6% (w/w) DHA, and a solid phase with 88.2% of SAT and monounsaturated fatty acid ethyl ester (MONOUNSAT), which could be used for biodiesel purposes after a hydrogenation step.

Crypthecodinium cohnii growth was studied on pure carbon sources (glucose, acetate, glycerol) and low-cost complex carbon sources (sugarcane molasses, crude glycerol and vinegar effluent) for lipid and DHA production. Among the pure substrates, glucose induced the highest lipid content (14.75% w/w DCW) and DHA content (7.14 mg g−1 DCW). Among the low-cost substrates, the highest lipid and DHA content were observed for the crude glycerol assay (14.7% w/w DCW and 6.56 mg g−1, respectively). Molasses induced the highest proportion of DHA of total fatty acids (49.58% w/w TFA) among all the substrates studied. Flow cytometric analysis revealed that the vinegar effluent induced the highest proportion of C. cohnii cells with injured membrane (92.8%). These results foresee the possibility of using these low-cost substrates at a larger scale for C. cohnii DHA and biodiesel production, aiming at zero wastes and process costs reduction. [ABSTRACT FROM AUTHOR]

• Organosolv is an efficient process to recover extractives and pectins from EOP. • Galacturonic acid and pectin can serve as carbon sources for microalgae C. cohnii. • DHA was accumulated up to 28% of total fatty acids using different EOP liquors. • A novel process for cost-effective valorization of EOP is suggested. Exhausted olive pomace (EOP) represents a potential candidate side stream to be utilized in biotechnological processes. EOP composition includes significant amounts of extractives and pectin, which are both usually discarded and are not utilized in the valorization process of the raw material. In this study, organosolv technology was optimized to remove the extractives and pectin using a Central Composite Rotatable Design. Optimal pretreatment conditions were predicted to be at 97.95 °C for 23.18 min, upon addition of 50% (v/v) EtOH in H 2 O, with 0.5% (w/v) of H 2 SO 4 as catalyst. The composition analysis of liquid fraction revealed a high content of total sugars (17.58 g/L), galacturonic acid (7.05 g/L) and phenolic compounds (2.97 g/L). The liquid fraction was utilized as a carbon source by the heterotrophic marine microalgae Crypthecodinium cohnii, where it was shown to promote lipid accumulation up to 38.5% wt. of cell biomass, even without any additional detoxification step. This study is the first report that shows the use of galacturonic acid as carbon source for the growth of C. cohnii , while underpinning the use of EOP as a promising substrate for the development of zero-waste bioprocesses. [ABSTRACT FROM AUTHOR]

Crypthecodinium cohnii is a marine heterotrophic dinoflagellate that can accumulate high amounts of omega-3 polyunsaturated fatty acids (PUFAs), and thus has the potential to replace conventional PUFAs production with eco-friendlier technology. So far, C. cohnii cultivation has been mainly carried out with the use of yeast extract (YE) as a nitrogen source. In the present study, alternative carbon and nitrogen sources were studied: the extraction ethanol (EE), remaining after lipid extraction, as a carbon source, and dinoflagellate extract (DE) from recycled algae biomass C. cohnii as a source of carbon, nitrogen, and vitamins. In mediums with glucose and DE, the highest specific biomass growth rate reached a maximum of 1.012 h−1, while the biomass yield from substrate reached 0.601 g·g−1. EE as the carbon source, in comparison to pure ethanol, showed good results in terms of stimulating the biomass growth rate (an 18.5% increase in specific biomass growth rate was observed). DE supplement to the EE-based mediums promoted both the biomass growth (the specific growth rate reached 0.701 h−1) and yield from the substrate (0.234 g·g−1). The FTIR spectroscopy data showed that mediums supplemented with EE or DE promoted the accumulation of PUFAs/docosahexaenoic acid (DHA), when compared to mediums containing glucose and commercial YE.

Dark fermentation is an anaerobic digestion process of biowaste, used to produce hydrogen as a fuel, which however releases high amounts of polluting volatile fatty acids in the environment. In order for the process to become more competitive, the acids stream can be utilized through conversion to high added-value docosahexaenoic acid by the microalga Crypthecodinium cohnii. Docosahexaenoic acid is one of the two main omega-3 fatty acids, necessary for human nutrition. The purpose of this work was to optimize the production of omega-3 fatty acids by the cells, utilizing the organic content of a dark fermentation effluent. For that purpose, the effect of different fermentation conditions was examined, such as incubation temperature, nitrogen source and concentration, the addition of chemical modulators, as well as the feeding composition. The volatile fatty acid content of the effluent was totally depleted in a fed-batch culture of the microalga, while the cells accumulated DHA in a percentage of 35.6% of total lipids, when fed with yeast extract or 34.2% when fed with ammonium sulfate. Taking into consideration the economic feasibility of the culture conditions proposed it was concluded that the use of yeast extract could be substituted by the much economic ammonium sulfate. • C. cohnii utilized VFA from a dark fermentation effluent for DHA production. • Fermentation growth medium was optimized for the efficient omega-3 production. • Substitution of commonly used yeast extract by ammonium sulfate as nitrogen source. • A two-stage fermentation protocol with a nitrogen limitation step was employed. [ABSTRACT FROM AUTHOR]

Background: Docosahexaenoic acid (DHA) is essential for human diet. However, high production cost of DHA using C. cohnii makes it currently less competitive commercially, which is mainly caused by low DHA productivity. In recent years, repeated fed-batch strategies have been evaluated for increasing the production of many fermentation products. The reduction in terms of stability of culture system was one of the major challenges for repeated fed-batch fermentation. However, the possible mechanisms responsible for the decreased stability of the culture system in the repeated fed-batch fermentation are so far less investigated, restricting the efforts to further improve the productivity. In this study, a repeated fed-batch strategy for DHA production using C. cohnii M-1-2 was evaluated to improve DHA productivity and reduce production cost, and then the underlying mechanisms related to the gradually decreased stability of the culture system in repeated fed-batch culture were explored through LC– and GC–MS metabolomic analyses. Results: It was discovered that glucose concentration at 15–27 g/L and 80% medium replacement ratio were suitable for the growth of C. cohnii M-1-2 during the repeated fed-batch culture. A four-cycle repeated fed-batch culture was successfully developed and assessed at the optimum cultivation parameters, resulting in increasing the total DHA productivity by 26.28% compared with the highest DHA productivity of 57.08 mg/L/h reported using C. cohnii, including the time required for preparing seed culture and fermentor. In addition, LC– and GC–MS metabolomics analyses showed that the gradually decreased nitrogen utilization capacity, and down-regulated glycolysis and TCA cycle were correlated with the decreased stability of the culture system during the long-time repeated fed-batch culture. At last, some biomarkers, such as Pyr, Cit, OXA, FUM, l-tryptophan, l-threonine, l-leucine, serotonin, and 4-guanidinobutyric acid, correlated with the stability of culture system of C. cohnii M-1-2 were identified. Conclusions: The study proved that repeated fed-batch cultivation was an efficient and energy-saving strategy for industrial production of DHA using C. cohnii, which could also be useful for cultivation of other microbes to improve productivity and reduce production cost. In addition, the mechanisms study at metabolite level can also be useful to further optimize production processes for C. cohnii and other microbes. [ABSTRACT FROM AUTHOR]

Docosahexaenoic acid (DHA) enriched in brain can yield many important degradation products after the attack of hydroxyl radicals, which is known to serve as a nutraceutical and neuroprotective effects. Oxidative stress is a commonly observed feature of Alzheimer's disease (AD). Therefore, uniformly radiolabeled DHA plays an important role in studying the oxidative fate of DHA in vivo and vitro. However, carbon isotope labeled DHA isn't commercially available now. The heterotrophic microalgae Crypthecodinium cohnii (C. cohnii) has been identified as a prolific producer of DHA. In this study, the growth rate and DHA production in C. cohnii were optimized in a new defined media, and the biosynthesis of U-13C-DHA from U-13C-glucose and U-14C-DHA from U-14C-glucose were analyzed by HPLC–MS/MS. Approximately 40 nmoles of U-13C-DHA with higher isotopic purity of 96.8% was produced in a 300 μL batch, and ~ 0.23 μCi of U-14C-DHA with significant specific activity of 5–6 Ci/mol was produced in a 300 μL batch. It was found that C. cohnii had the optimal growth and DHA accumulation at 25 °C in this defined media (C/N = 10). An efficient protocol for the biosynthesis of U-13C-DHA and U-14C-DHA were set up firstly, which provides the basic support for the analysis of oxidative degradation products of DHA in AD. [ABSTRACT FROM AUTHOR]

In the last decades, several plant-based materials were used for the substitution of fish meal and oil in aquaculture. The present study evaluated the fish quality and the sensory differences of rainbow trout (Oncorhynchus mykiss) and pike-perch (Sander lucioperca) from three different feeding groups, which were fed a commercially available industrial (standard) diet, a control diet, and a special microorganism-based feed mix. This feed mainly consisted of a mix made of Rhodotorula glutinis, Crypthecodinium cohnii, and Arthrospira sp. and had 50% less fish meal and fish oil compared to typical control diets. At the beginning, the pike-perch population was six months old, and the rainbow trout population was 15 months old. The feeding study duration was 16 weeks and every four weeks the growth performance and several morphometric parameters were recorded. Afterwards, sensory evaluation took place to identify possible trends. Sensory evaluation revealed that the rainbow trout groups did not show any significant differences to the standard and control fish fillets with regard to odor, texture, and taste. The effects on rainbow trout growth performances and carcass parameters were similar to the standard group. The feed mix was not optimal for pike-perch farming, which was also reflected by significantly adversely affected growth performance and carcass parameters. The sensorial evaluation showed an opposite trend: here, only small differences in the fillets from the feed mix and standard/control diet were observed.

Abstract The effect of varying concentrations of the nitrogen source on the growth kinetics, lipid accumulation, lipid and DHA productivity, and fatty acid composition of C. cohnii was elucidated. Growth of C. cohnii was in three distinct growth stages: cell growth, lipid accumulation and a final lipid turnover stage. Most of lipids were accumulated in lipid accumulation stage (48–120 h) though, slow growth rate was observed during this stage. NaNO3 supported significantly higher lipid content (26.9% of DCW), DHA content (0.99 g/L) and DHA yield (44.2 mg/g glucose) which were 2.5 to 3.3-folds higher than other N-sources. The maximum level of C16–C18 content (% TFA) was calculated as 43, 54 and 43% in lipid accumulation stage under low nitrogen (LN, 0.2 g/L), medium nitrogen (MN, 0.8 g/L) and high nitrogen (HN, 1.6 g/L) treatments, respectively. Cultures with LN, by down-regulating cell metabolism, trigger onset of lipogenic enzymes. Conversely, NAD+/NADP+-dependent isocitrate dehydrogenase (NAD+/NADP+-ICDH) were less active in LN than HN treatments which resulted in retardation of Kreb’s Cycle and thereby divert citrate into cytoplasm as substrate for ATP-citrate lyase (ACL). Thereby, ACL and fatty acid synthase (FAS) were most active in lipid accumulation stage at LN treatments. Glucose-6-phosphate dehydrogenase (G6PDH) was more active than malic enzyme (ME) in lipid accumulation stage and showed higher activities in NaNO3 than other N-sources. This represents that G6PDH contributes more NADPH than ME in C. cohnii. However, G6PDH and ME together seems to play a dual role in offering NADPH for lipid biosynthesis. This concept of ME together with G6PD in offering NADPH for lipogenesis might be novel in this alga and needed to be explored.

Highlights • A method for valorizing by-products of a biohydrogen production process is proposed. • Microalga Crypthecodinium cohnii is cultivated on a dark fermentation effluent. • Volatile fatty acids of the effluent were efficiently bioconverted into omega-3. • Cells grown on effluent reached an omega-3 content of 29.8% of total fatty acids. Abstract Dark fermentation is an anaerobic digestion process of biowaste, used to produce hydrogen- for generation of energy- that however releases high amounts of polluting volatile fatty acids, such as acetic acid, in the environment. In order for this biohydrogen production process to become more competitive, the volatile fatty acids stream can be utilized through conversion to high added-value metabolites, such as omega-3 fatty acids. The docosahexaenoic acid is one of the two most known omega-3 fatty acids and has been found to be necessary for a healthy brain and proper cardiovascular function. The main source is currently fish, which obtain the fatty acid from the primary producers, microalgae, through the food chain. Crypthecodinium cohnii , a heterotrophic marine microalga, is known for accumulating high amounts of docosahexaenoic acid, while offering the advantage of assimilating various carbon sources, such as glucose, ethanol, glycerol and acetic acid. The purpose of this work was to examine the ability of a C. cohnii strain to grow on different volatile fatty acids, as well as, on a pretreated dark fermentation effluent and accumulate omega-3. The strain was found to grow well on relatively high concentrations of acetic, butyric or propionic acid as main carbon source in a fed-batch pH-auxostat. Most importantly, C. cohnii totally depleted the organic acid content of an ultra-filtrated dark fermentation effluent after 60 h of fed-batch cultivation, therefore offering a bioprocess not only able to mitigate environmental pollutants, but also to provide a solution for a sustainable energy production process. The accumulated docosahexaenoic acid content was as high as 29.8% (w/w) of total fatty acids. [ABSTRACT FROM AUTHOR]

Highly pure docosahexaenoic acid (DHA) was produced successfully in a packed bed reactor via the two-step lipase-catalysed esterification using the fatty acid from microalgae (from Crypthecodinium cohnii) and ethanol as substrates. Lipozyme RM IM from Rhizomucor miehei was employed as a biocatalyst. In the first step, a temperature of 40 °C, a molar ratio of 1:4 (fatty acid to ethanol), and a water content of 0.6 wt% (based on the total substrate weight) were selected as optimum conditions. A maximum DHA concentration of ca. 90 wt% was achieved in the fatty acid fraction with an 89 wt% yield after 15 min. In the second step, the fatty acids from the first step were used as the substrate and a recursive reaction was conducted using identical optimized conditions. Following this second step, a DHA concentration of 100 wt% was achieved in conjunction with an 83.2 wt% yield after 25 min.

The heterotrophic microalga Crypthecodinium cohnii has attracted considerable attention due to its capability of accumulating lipids with a high fraction of docosahexaenoic acid (DHA). In our previous study, ethanolamine (ETA) was identified as an effective chemical modulator for lipid accumulation in C. cohnii. In this study, to gain a better understanding of the lipid metabolism and mechanism for the positive effects of modulator ETA, metabolic flux analysis was performed using 13C-labeled glucose with and without 1 mM ETA modulator. The analysis of flux distribution showed that with the addition of ETA, flux in glycolysis pathway and citrate pyruvate cycle was strengthened while flux in pentose phosphate pathway was decreased. In addition, flux in TCA cycle was slightly decreased compared with the control without ETA. The enzyme activity of malic enzyme (ME) was significantly increased, suggesting that NADP+-dependent ME might be the major source of NADPH for lipid accumulation. The flux information obtained by this study could be valuable for the further efforts in improving lipid accumulation and DHA production in C. cohnii.

Extracellular polysaccharides (EPS) produced by marine microalgae have the potential to be used as antioxidants, antiviral agents, immunomodulators, and anti-inflammatory agents. Although the marine microalga Crypthecodinium cohnii releases EPS during the process of docosahexaenoic acid (DHA) production, the yield of EPS remains relatively low. To improve the EPS production, a novel mutagenesis of C. cohnii was conducted by atmospheric and room temperature plasma (ARTP). Of the 12 mutants obtained, 10 mutants exhibited significantly enhanced EPS yield on biomass as compared with the wild type strain. Among them, mutant M7 was the best as it could produce an EPS volumetric yield of 1.02 g/L, EPS yield on biomass of 0.39 g/g and EPS yield on glucose of 94 mg/g, which were 33.85%, 85.35% and 57.17% higher than that of the wild type strain, respectively. Results of the present study indicated that mutagenesis of the marine microalga C. cohnii by ARTP was highly effective leading to the high-yield production of EPS.

In this study, date syrup was used for the first time as an alternative carbon source for Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) production by response surface methodology. Four medium variables including date syrup concentration, pH, yeast extract and sea salt concentrations were considered to optimize biomass production. Maximum biomass concentration, 6.6 g/L, was achieved in 17 g/L date syrup concentration, 2 g/L yeast extract, 20 g/L sea salt and pH 7. After this optimization, a two-step feed strategy in fed batch cultivation was done in the optimized date based medium to enhance DHA production. The first step was dedicated to biomass formation and the second step was designed for DHA production. In the second step, single and for the first time, the combined effects of growth temperature shift from 25 to 20 °C, and nitrogen starvation on DHA production were investigated. The results revealed that the most appropriate strategy was a fed batch cultivation combined with both nitrogen depletion and temperature growth shift after 6th days from inoculation, leading to an enhancement of DHA production to 5.65 g/L, which was approximately twice the amount obtained considering the fact that the control was 2.9 g/L. Results revealed that date syrup based medium with two-step fed batch cultivation under combined stress can be an effective strategy for the production of valuable microalgal metabolites in heterotrophic conditions. [ABSTRACT FROM AUTHOR]

Oxygen supply is an important factor during Crypthecodinium cohnii fermentation for docosahexaenoic acid (DHA) production. However, few studies about the intrinsic correlation between dissolved oxygen (DO) and cellular metabolism have been reported. In this study, the responses of C. cohnii to different DO levels were evaluated. The results showed the growth and glucose consumption rates of C. cohnii were much higher under high oxygen supply condition. Furthermore, GC-MS based comparative metabolomic analysis was employed to discriminate the responsive metabolites associated with varying DO levels. The results showed the intermediates involved in glycolytic pathway and TCA cycle were up-regulated under high DO levels at exponential phase. At stationary phase, under high DO levels, metabolites involved in triacylglycerol metabolism were up-regulated, while the OPP pathway intermediate product ribose 5-phosphate was down-regulated. Together, these results provide useful insights into the functional metabolic relationship between DO levels and DHA production in C. cohnii . [ABSTRACT FROM AUTHOR]

The heterotrophic microalga Crypthecodinium cohnii has attracted considerable attention due to its capability of accumulating lipids with a high fraction of docosahexaenoic acid (DHA). In our previous study, ethanolamine (ETA) was identified as an effective chemical modulator for lipid accumulation in C. cohnii. In this study, to gain a better understanding of the lipid metabolism and mechanism for the positive effects of modulator ETA, metabolic flux analysis was performed using 13C-labeled glucose with and without 1 mM ETA modulator. The analysis of flux distribution showed that with the addition of ETA, flux in glycolysis pathway and citrate pyruvate cycle was strengthened while flux in pentose phosphate pathway was decreased. In addition, flux in TCA cycle was slightly decreased compared with the control without ETA. The enzyme activity of malic enzyme (ME) was significantly increased, suggesting that NADPC- dependent ME might be the major source of NADPH for lipid accumulation. The flux information obtained by this study could be valuable for the further efforts in improving lipid accumulation and DHA production in C. cohnii. [ABSTRACT FROM AUTHOR]

Fishmeal and oil are regarded as byproducts resulting from fish processing. However, the procedures are hypothesized to be an ecological harm for maintaining the balance of the aquatic ecosystems. This study aimed at characterizing an innovative algae-yeast mix for a partial substitution of fishmeal and oil in fish feed. The microorganism mix consisted of the cyanobacterium Arthrospira sp., the microalgae Crypthecodinium cohnii, and the yeast Rhodotorula glutinis to create a balanced nutritional composition with an enrichment of bioactive compounds for inducing positive health effects in selected fish species. Such a modification can also be interpreted in terms of a positive (human) nutritional effect. From an ecological point of view, all these microorganisms can be cultivated without minimum effort. In fish feeding trials, pike-perch ( Sander lucioperca) and rainbow trout ( Oncorhynchus mykiss) were taken for evaluating the feed acceptance, fish health, and fish filet quality. The study revealed a successful feed development, which resulted in healthy organisms depending on the species and compared to conventionally fed organisms. [ABSTRACT FROM AUTHOR]

Exopolysaccharide from Crypthecodinium cohnii (EPCP1-2) is a marine exopolysaccharide that evidences a variety of biological activities. We isolated a neutral polysaccharide from the fermentation liquid of Crypthecodinium cohnii (CP). In this study, a polysaccharide that is derived from Crypthecodinium cohnii were analyzed and its anti-inflammatory effect was evaluated on protein expression of toll-like receptor 4 and nuclear factor κB pathways in macrophages. The structural characteristics of EPCP1-2 were characterized by GC (gas chromatography) and GC-MS (gas Chromatography-Mass Spectrometer) analyses. The molecular weight was about 82.5 kDa. The main chain of EPCP1-2 consisted of (1→6)-linked mannopyranosyl, (1→6)-linked glucopyranosyl, branched-chain consisted of (1→3,6)-linked galactopyranosyl and terminal consisted of t-L-Rhapyranosyl. The in vitro anti-inflammatory activity was representated through assay of proliferation rate, pro-inflammatory factor (NO) and expressions of proteins on RAW 264.7, the macrophage cell line. The results revealed that EPCP1-2 exhibited significant anti-inflammatory activity by regulating the expression of toll-like receptor 4, mitogen-activated protein kinases, and Nuclear Factor-κB protein. [ABSTRACT FROM AUTHOR]

In the present work, a novel OxiOrganosolv pretreatment was developed for the fractionation of beechwood. Τhe process employs a mixture of water and common organic solvents, polyoxometalate complexes (POMs) as homogeneous catalysts and O 2 that acts as an oxidative agent enabling their regeneration. Due to their strong oxidizing ability and redox reversibility, POMs can achieve efficient biomass fractionation by catalyzing simultaneously hemicellulose hydrolysis and oxidation of lignin ether bonds that leads to its depolymerization, dissolution in the organic solvent and removal from the cellulose-rich pulp. According to the results, both commercially available phosphomolybdic acid (HPMo) and also the in-house produced Fe- and Cu- exchanged salts successfully promoted delignification and hemicellulose removal compared to the OxiOrganosolv pretreatment in the absence of catalyst. Most importantly, catalytic lignin depolymerization was achieved without the use of mineral acids that are typically used in a traditional organosolv. Evaluation of the enzymatic digestibility of the pulps revealed HPMo as the most favorable catalyst for increased saccharification yields and production of sugar-rich hydrolysates that were further used as carbon sources for fermentation and production of ω -3 polyunsaturated fatty acids (ω -3 PUFAs) from the heterotrophic microalgae Crypthecodinium cohnii. Hydrolysates produced from HPMo and Fe-PMo-assisted OxiOrganosolv pretreatment supported cell growth and led to high accumulation of ω -3 PUFAs, while severe inhibition was observed with the hydrolysates from Cu-PMo pretreated biomass. This study demonstrates the possibility of using POMs as catalysts for efficient biomass delignification with the aim to obtain sugar-rich streams that can be utilized for the production of nutraceuticals. • A novel OxiOrganosolv process with POMs as homogeneous catalysts was developed. • POMs led to efficient delignification and hemicellulose removal from beechwood. • HPMo was the best catalyst to increase cellulose digestibility and glucose yields. • HPMo and Fe-PMo hydrolysates promoted ω3 accumulation in microalgae C. cohnii. • The presence of Cu in Cu-PMo hydrolysates significantly hindered microbial growth. [ABSTRACT FROM AUTHOR]

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Many different wastewater and by-products derived from industrial activities potentially support microalgal growth by providing a cost-effective and sustainable solutions. In this present study, it was aimed to biologically utilize cheese whey (CW) and corn steep liquor (CSL) for microalgal biomass and lipid production by using these wastes in culture media for heterotrophic microalga Crypthecodinium cohnii cultivation. To determine nutrient requirements for C. cohnii growing in a medium prepared with CSL and in CW, statistical screening tools were used. CSL significantly enhanced microalgal growth and it could be an alternative to yeast extract as the primary nutrient source. As for CW, it served as a good culture medium for C. cohnii with the supplement of some of nutrients and eliminated the need for fresh water. Thus, a new culture medium was developed by combining undiluted CW and CSL and optimized for the growth of C. cohnii. Lastly, in a scale-up attempt by using this new medium, microalgal production was performed in a 3 L stirred tank bioreactor. C. cohnii yielded relatively high biomass productivity (2.28 g/L.d) and lipid content (28.7% dry weight) in the optimized medium. Although C. cohnii was known for its ability to accumulate high amounts of docosahexaenoic acid (DHA), it transformed its fatty acid composition to an increased proportion of saturated and monounsaturated fatty acids (C16:0-C18:1) that comprise ~70% of total fatty acids (TFA) when it was cultivated in CW mainly supplemented with CSL. Thus, C. cohnii seemed to be more feasible for biodiesel production than any other purposes when it was cultivated in this new medium. [ABSTRACT FROM AUTHOR]

The effect of varying concentrations of the nitrogen source on the growth kinetics, lipid accumulation, lipid and DHA productivity, and fatty acid composition of C. cohnii was elucidated. Growth of C. cohnii was in three distinct growth stages: cell growth, lipid accumulation and a final lipid turnover stage. Most of lipids were accumulated in lipid accumulation stage (48-120 h) though, slow growth rate was observed during this stage. NaNO supported significantly higher lipid content (26.9% of DCW), DHA content (0.99 g/L) and DHA yield (44.2 mg/g glucose) which were 2.5 to 3.3-folds higher than other N-sources. The maximum level of C16-C18 content (% TFA) was calculated as 43, 54 and 43% in lipid accumulation stage under low nitrogen (LN, 0.2 g/L), medium nitrogen (MN, 0.8 g/L) and high nitrogen (HN, 1.6 g/L) treatments, respectively. Cultures with LN, by down-regulating cell metabolism, trigger onset of lipogenic enzymes. Conversely, NAD/NADP-dependent isocitrate dehydrogenase (NAD/NADP-ICDH) were less active in LN than HN treatments which resulted in retardation of Kreb's Cycle and thereby divert citrate into cytoplasm as substrate for ATP-citrate lyase (ACL). Thereby, ACL and fatty acid synthase (FAS) were most active in lipid accumulation stage at LN treatments. Glucose-6-phosphate dehydrogenase (G6PDH) was more active than malic enzyme (ME) in lipid accumulation stage and showed higher activities in NaNO than other N-sources. This represents that G6PDH contributes more NADPH than ME in C. cohnii. However, G6PDH and ME together seems to play a dual role in offering NADPH for lipid biosynthesis. This concept of ME together with G6PD in offering NADPH for lipogenesis might be novel in this alga and needed to be explored. [ABSTRACT FROM AUTHOR]

Crypthecodinium cohnii is a heterotrophic dinoflagellate that can achieve a lipid content greater than 20% on dry weight. DHA can represent up to 40% of the total fatty acids. Our objective was to investigate the variation in fatty acid content when growth conditions are optimal compared with when there is limited growth, due to the absence of nitrogen in the media. Nitrogen-limited growth conditions caused the cells to stop dividing and accumulate lipids, principally as docohexanoic acid (DHA). Oxygen availability in the culture favoured DHA accumulation. In the cultures without nitrogen and with oxygen availability there was a lipid cell content 3,18 times higher than in the control condition (with nitrogen and air), and 2,25 higher than those without nitrogen and air. C. cohnii can be used as an optimal DHA source for the production of phytodiets in aquaculture.